The Effect Of Melanoma Antigen A3Silence On The Proliferation, Apoptosis And Invasion In Hepatocellular Carcinoma

Objective: Melanoma antigen gene A3(MAGE-A3) aberrantly expresses in a numberof cancer types and has been used extensively as a target for anti-tumor vaccines.However, its function is unclear. The melanoma antigen A3siRNA interferencefragments were synthesized by a chemical process, The aim of this study wastransfect HepG2cells and Huh-7cells. To identify the effect on the progression,apoptosis, metastasis and invasion in hepatocellular carcinoma (HCC) andpreliminary elucidate its molecular mechanisms while MAGE-A3gene wasknockdown by RNA interference.Methods:(1) The expression of MAGE-A3gene of HepG2, Huh7, Hep3B andSK-Hep1was detected by Q-PCR, the melanoma antigen A3siRNA interferencefragments were synthesized by a chemical process, followed by transfecting HepG2cells and Huh-7cells. The experiment were divided idling group (Only plustransfection reagent), negative control group and MAGE-A3siRNA interferencegroup (interference Group). The transfection efficiency was detected by Invertedfluorescence microscope. Detect the gene and protein expression of MAGE-A3by Q-PCR and Western-Blot technology after siRNA transfection.(2) The growth andproliferation of HepG2and Huh-7cells were detected by MTT assays, the migrationand invasion were detected by transwell technique.(3) The cell cycle and apoptosis ofHepG2and Huh-7cells in each group were analysis by Flow cytometry(FCM) assays.(4) The gene and protein expression of p53, bcl-2, survivin and bax in HepG2cellswere detected by Q-PCR and Western Blot in hepatocellular carcinoma cells afterMAGE-A3silence.Results:1. The mRNA level of MAGE-A3gene was high in hepatocellular carcinoma celllines HepG2and Huh-7compared to Hep3B and SK-Hep1.2. After transfecting MAGE-A3siRNA fragments to HepG2and Huh-7cells for 4-6h, green fluorescence was observed by inverted fluorescence microscope. Thetransfection efficiency was80%after transfection for24h, and48h later, thetransfection efficiency was95%.3. The screening of MAGE-A3siRNA effective fragment: After transfected withMAGE-A3siRNA664fragments in HepG2and Huh7cells for48h, the mRNAlevel of MAGE-A3was knockdowned more than70%by Q-PCR assay. Theexpression of MAGE-A3protein was significantly reduced in the two cells linesby western blot test.4. After transfected with MAGE-A3siRNA for24h,48h,72h,96h,120h, there wasno significant difference in the proliferation of HepG2and Huh-7cell linesbetween MAGE-A3siRNA group and controls by MTT assay.5. The transwell technique show, after the silence of MAGE-A3, the migration cellsof interference group in HepG2and Huh-7were42.8±7.12and21±1.78, theinvasion cells of interference group in HepG2and Huh-7were35.2±4.32and18±3.18, that significantly lower than the negative control group and idling group,the difference was statistically significant (P<0.05). It shown the silence ofMAGE-A3can inhabit migration and invasion of HepG2cells and Huh7cells.6. FCM analysis confirmed, the silence of MAGE-A3can affect the cell cycle ofHepG2and Huh7, the ratio of cells in G2phase were increased in tow cell lines.It show that the silence of the MAGE-A3lead to cells G2arrest in HepG2cellsand Huh7cells. And the apoptosis of hepatoma cells was detect by annexin V, theresults showed that the HepG2and Huh7apoptotic cells were increased, butHepG2cells significantly.7. The gene and protein expression of p53, bcl-2, survivin and bax in HepG2cellswere detected by Q-PCR and Western Blot. The results showed that p53and baxexpression increased, while bcl-2and survivin expression decreased whenMAGE-A3gene was knockdown. It suggested that MAGE-A3silence may beinduced by p53-dependent apoptosis pathway.Conclution:1. The siRNA segment of MAGE-A3gene was identified, which may significantly knockdown the level of MAGE-A3gene and protein in human HepG2and Huh-7cell lines.2. Although there is no significant effect on the growth and proliferation of HepG2and Huh-7cell lines after MAGE-A3silence, the ability of cell migration andinvasion significantly decreases, and the number of apoptotic cell increases.3. The molecular mechanism of cell invasion and apoptosis induced by MAGE-A3gene silence in hepatocellular carcinoma may be related to p53-dependentapoptosis pathway.