Effects Of TIMP-3Gene On Proliferation, Apoptosis, Invasiveness And Metastasis Of Human Hepatocarcinoma Cell Lines

ObjectiveTo construct a eukaryotic mediation vector with stable TIMP-3geneover-expression and transfect it into SMMC-7721and HepG2cell lines forinvestigating its effects on proliferation, apoptosis, cell cycle, invasiveness andmigration of these hepatocellular(HCC) cells.Methods1. The amplification and identification of the plasmid pCMV6-AC-GFP/TIMP-3(TIMP-3) and pCMV6-AC-GFP (negative control, NC): the diluted plasmid TIMP-3and plasmid NC were transferred into Escherichia coli. After collecting bacteria liquidand sequencing, the plasmids were obtained purely without TIMP-3and NCendotoxins for subsequent experiments.2. The amplified plasmid TIMP-3and NC were respectively transfected into thetwo HCC cell lines via the non-liposome transfection reagent. As the control groupcells died in2weeks of G418800μg/ml screening, lower G418concentration(400μg/ml) for2weeks was tried. Positive single cell clones were successfullyestablished. Their expressions of TIMP-3protein were detected by Western Blot.3. Cell growth curves were drawn by MTS experiments to detect the growth of theHCC cells after the stable expression of TIMP-3.4. The apoptosis and cell cycle assays were performed by flow cytometry after the stable expression of TIMP-3.5. The invasiveness and migration assays were performed using Transwellchambers to detect the invasiveness and migration of the two HCC cells after thestable expression of TIMP-3.Results1. TIMP-3and NC plasmid were successfully transferred into Escherichia coli. Theresults were consistent with the original sequence.Therefore they were qualified forsubsequent experiments.2. The TIMP-3and NC plasmids were transfected into SMMC-7721and HepG2cells respectively. The monoclonal positive cells expressed high level of TIMP-3stably. The positive single cell clones were named SMMC-7721and HepG2cells inTIMP-3group, whereas those containing the empty vector were named SMMC-7721and HepG2cells in NC group.Western blot showed that the cells in TIMP-3groupexpressed TIMP-3protein, while those in NC group did not.3. MTS assay demonstrated that proliferation of HCC cells transfected withTIMP-3was significantly suppressed compared to that of control (in day5, P=0.003and P=0.009respectively).4. Flow cytometry showed that in the two HCC cells, TIMP-3induced apoptosis[(SMMC-7721:30.7±1.6%vs8.4±1.1%, P=0.001; HepG2:12.6±1.1%vs7.6±1.3%,P=0.038)], and the resulted in cell cycle arrest at G2/M phase [SMMC-7721:G0/G1:(36.075±3.53)%vs (57.72±1.53)%; G2/M:(51.91±1.71)%vs (30.11±0.20)%, P=0.030;HepG2: G0/G1:(49.82±1.46)%vs (59.47±1.27)%,G2/M:(26.27±0.54)%vs(14.15±3.6)%, P=0.034].5. Invasiveness and migration assay indicated that TIMP-3significantlydecreased the invasion and migration of the two HCC cells [invasiveness assay:(SMMC-7721:60±11vs121±17, P=0.033; HepG2:98±6vs192±22, P=0.015);migration assay:(SMMC-7721:53±8vs102±10, P=0.038; HepG2:109±9vs232±13,P=0.001)]. Conclusion1. Two HCC cell lines (SMMC-7721and HepG2) with stable expression ofTIMP-3protein were successfully constructed via stable transfection.2. TIMP-3gene significantly inhibited the proliferation, invasion and migrationabilities of HCC cells, and induce apoptosis and G2/M phase arrest.