Effects Of Knockdown Of MMP2 On The Proliferation,Apoptosis And Invasion Of Human Hepatoma Cells Incubated With Conditioned Medium

ObjectiveTo suppress the MMP2 gene in hepatocellular carcinoma cell lines HepG2 that are moderately differentiated and Huh-7 that are highly differentiated by usingRNA interference technology and to explore the effects on the proliferation,apoptosis and invasion of hepatoma cells induced by conditioned medium derived from highly metastatic hepatoma cells MHCC-97 H,which provides a theoretical basis for further research on the role of liver cancer cell heterogeneity in the invasion and metastasis mechanism of liver cancer.MethodsMMP2-shRNA eukaryotic expression plasmid was designed,then transfected it into HepG2 and Huh-7 cell lines with liposomes.The expressions of MMP2 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blot.To clarify the inhibitory effect of MMP2-shRNA,MMP2 enzyme activity was detected by gelatin zymography.Culture supernatant of MHCC-97 H cells was collected as conditioned medium.It was used to incubate HepG2 cells which were transfected or not transfected with MMP2-shRNA for 48 h.And it was used to incubate Huh-7 cells which were transfected or not transfected with MMP2-shRNA for 72 h.Then MTT was used to detect the cell proliferation ability.Using flow cytometry to detect the effect of decreasing MMP2 gene expression on the apoptosis of HepG2 and Huh-7 cells.The transwell invasion experiments were used to observe the invasion ability of hepatoma cells HepG2 and Huh-7.ResultsAfter transient transfecting the designed MMP2-shRNA into HepG2 and Huh-7cells,MMP2 inhibition effects were detected in different time points.The results of quantitative PCR and Western blot showed the expression of MMP2 mRNA and protein in both cells were significantly down-regulated.The lowest expression of MMP2 mRNAand protein was detected after 48 hours for HepG2 cells and was detected after 72 hours for Huh-7 cells.Gelatin zymogram analysis confirmed that MMP2 activity was inhibited in both cells after transfection.The MTT test showed that inhibiting the expression of the MMP2 gene can inhibit the proliferation of the HepG2 and Huh-7 cells incubated by the conditioned medium.It was found that knocking down the MMP2 gene can cause a significant increase in the apoptosis rate of HepG2 and Huh-7 cells cultured in conventional and conditioned medium,as determined by flow cytometry.The Transwell invasion experiment proved that,compared with the conventional medium,the invasion ability of the HepG2 and Huh-7 cells in the conditioned medium was enhanced.After knocking down the MMP2 gene,the invasion ability of the cells in both mediums was correspondingly weakened.ConclusionCompared with the conventional medium,conditioned medium derived from MHCC-97 H cells can significantly induce the proliferation and invasion of HepG2 and Huh-7 cells,and reduce the apoptosis of the cells.It indicates that highly metastatic hepatoma cells may induce the malignant transformation of adjacent hepatoma cells through paracrine actio.Knocking down the expression of MMP2 gene in HepG2 and Huh-7 cells can significantly inhibit the above effects of conditioned medium.Therefore,the MMP2 gene has an important effect on the proliferation,apoptosis,and invasion ability of liver cancer cell lines,and can be used as an important target for liver cancer targeted therapy.