Inhibition Of Invasiveness Of Pancreatic Carcinoma Cell Line PANC-1 By Suppression Of MMP-2 Gene Expression Using RNA Interference

Objective To investigate the expression of matrix metalloproteinases-2 (MMP-2) protein and unravel its clinical significance in pancreatic carcinoma. To construct MMP-2 small interfering RNA (siRNA) plasmid vector and investigate the inhibitory effect of RNA interference(RNAi) on MMP-2 gene expression and protein level of PANC-1 cell as well as cell invasiveness, to lay the foundation for further investigation on the invasion mechanisms of pancreatic carcinoma and for the development of strategy of anti- invasion therapy in pancreatic carcinoma.Methods The immunohistochemical method was used to determine the expression of MMP-2 in 36 paraffin embedded samples of pancreatic carcinoma, compared with that in normal pancreas tissues of 14 cases, then revealed the relationship between MMP-2 expression and clinical features of pancreatic carcinoma. SiRNA for MMP-2 gene was designed. Two single-strand DNA templates of small hairpin RNA (shRNA) were synthesized according to the sequence of the siRNA. After the DNA templates synthesized, two different restriction sites of BamHâ… and Hindâ…¢were superimposed, respectively, to the two ends of it. The insertion element formed after the DNA templates annealed. Make the blank plasmid linearization using restriction enzymes, then the insertion element was inserted into the blank plasmid by T4 ligase. PCR and sequencing were performed to check whether MMP-2 siRNA plasmid be constructed successfully or not. MMP-2 siRNA plasmid and negative control plasmid were transfected to pancreatic carcinoma cell line PANC-1 cells with Lipofectamine? 2000. As a reporter gene, green fluorescence protein(GFP) was evaluated by flow cytometry and fluorescent microscopy to estimate the efficiency of transfection. Then positive cell monoclone was selected and cultivated with G418 for 8 weeks. RQ-PCR was used to detect the expression of MMP-2 mRNA. The expression of MMP-2 protein was determined by enzyme-linked immunosorbent assay (ELISA) and immunocyto- chemistry. The influence on invasion ability of PANC-1 cells after RNA interference was measured by transwell chamber experiment. MTT assay was used to detect the proliferation and growth of PANC-1 cells.Results (1) The positive percentage of MMP-2 protein expression was 66.7% in pancreatic carcinoma, compared with 14.3% in normal pancreatic tissue, presenting statistical significance between the two groups. The expression levels of MMP-2 were obviously different in clinical stages, but had nothing to do with differentiation of pancreatic carcinoma. (2) PCR and sequencing confirmed that the MMP-2 siRNA plasmid was successfully constructed. (3) After positive cell monoclone was selected with G418, the best efficiency of transfecting recombinant plasmid was 82.1%. (4) When transfected by MMP-2 siRNA plasmid, the MMP-2 gene expression of PANC-1 was effectively suppressed and the suppression rate was 71.74%,and the suppression rate of MMP-2 protein was reached to 49.82%. By immunocytochemical stain, MMP-2 was strongly positive stained in the control group, while weekly positive stained in the group transfected by the MMP-2 siRNA plasmid. (5) With transwell chamber experiment, the inhibition ratio of invasiveness of the group transfected by the MMP-2 siRNA plasmid was 33.0%. Compared with controls,the group transfected by the recombinant plasmid had no difference in proliferation rate measured by MTT assay(P>0.05).Conclusions The expression of MMP-2 is potentialized in pancreatic carcinoma, and it is positively correlated with the clinical stages or lymph node metastasis of pancreatic carcinoma, but have nothing to do with cancer differentiation. The MMP-2 siRNA plasmid vector is successfully constructed in vitro and efficiently transfected to PANC-1 cells. MMP-2 siRNA plasmid vector can effectively suppress MMP-2 mRNA and protein expression in human pancreatic carcinoma cell line PANC-1. At the same time, the invasiveness of PANC-1 cells decreases obviously. All these suggests that MMP-2 gene could be a target for anti-invasion therapy of pancreatic carcinoma, RNAi is expected to be an effective measure for tumor gene therapy.