Antisense Oligodeoxynucleotide Targeting MMP7 Influences Proliferation, Sensitivity Of Apoptosis And Invasion In Pneumonic Adenocarcinoma Cell Line A549

Background and objectiveMatrilysin(MMP7) is a kind of Zn-depended proteolytic enzyme. It was found to be expressed in various tumor cells including human pneumonic adenocarcinoma. MMP7 was found to be involved in cleaving extracellular matrix(ECM) and basement membrane(BM), enhancing invasion of tumor cells, inhibiting sensitivity of apoptosis. MMP7 was found to highly express on nonsmall cell lung cancer in our previous research. The aim of this study is to observe the proliferation and apoptosis and invasion of tumor cell in vitro through inhibiting the expression of MMP7 by antisense gene technology.Methods①MMP7 antisense oligodeoxynucleotide(ASODN) targeting MMP7 mRNA was designed, constructed, and lipofected into cell line A549.②Compared with A549 group and SCODN group, the expression of MMP7 mRNA and protein was examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry.③The ability of proliferation was oberseved in ASODN group, SCODN group and control group cells respectively by MTT method. With or without MMP7 ASODN existence, cell shape was observed by inverted microscope and electronm- icroscope. Cell cycle was examined by Flow Cytometer.④After MMP7 ASODN was tranfected, the expression of Fas was examined by immunohistochemistry and Flow Cytometer. The rates of apoptosis were compared in three groups afer the FasL was added.⑤With or without MMP7 ASODN existence, the ability of adhesion and the ability of invasion were compared in plate adhesion model and Boyden chamber model.Results①Compared with SCODN group and control group, MMP7 mRNA expression was downregulated by RT-PCR method markedly in ASODN group (P<0.01). MMP7 protein was also downregulated by immunohistochemistry.②The proliferation of A549 cells was inhibited by MMP7 ASODN of different concentration by MTT method(P<0.05). The 5μmol/L ASODN group had no apparent difference compared with the 7.5μmol/L ASODN group(P>0.05). The inhibition peaked at 48 hour. After A549 cells were transfected with MMP7 ASODN, the volume and number of these cells decreased. Dropsy was found in mitochondrion and endoplasmic reticulum by electronmicroscope. And MMP7 ASODN transfection could inhibit the transition period of G0/G1 phase to S phase.③After MMP7 ASODN was tranfected, the expression of Fas was upregulated by immunohistochemistry and Flow Cytometer. The rate of apoptosis in ASODN group was increased compared with SCODN group and control group after the FasL was added(P<0.01).④The ability of adhesion in ASODN group was decreased compared with control group (P<0.05). Also the ability of invasion in ASODN group was decreased compared with control group (P<0.01).Conclusions①The expression of MMP7 mRNA and MMP7 protein could be decreased successfully by MMP7 ASODN.②The MMP7 ASODN transfection could inhibit the ability of proliferation and the transition period of G0/G1 phase to S phase.③The ASODN could upregulate the expression of Fas and increase the sensitivity of apoptosis of A549 cells.④The ability of adhesion and the ability of invasion of A549 cells were inhibited by MMP7 ASODN .