| ObjectiveHepatocellsular carcinoma(HCC) is a common tumor of high malignant and poor prognosis. Surgical removal is the most effective treatment way in early liver cancer, but chemotherapy drugs is still an important auxiliary treatment approaches for the middle- late HCC patients. The melanoma antigen gene(MAGE) in normal tissues is unexpressed except testis and placenta, but is highly expressed in various tumor, and MAGE is related to the invasion, metastasis, recurrence, prognosis and drug-resistant of malignant tumor. The melanoma antigen A3 si RNA interference fragments were synthesized by a chemical process. The aim of this study was transfect human liver cells lines Hep G2 and Huh-7,then we add the chemotherapy drugs treatment, to observate the effect of MAGE-A3 on the sensitivity to chemotherapy drugs of hepatocellsular carcinoma cells Hep G2 and Huh-7 after inhibited by RNAi, and preliminary elucidate its molecular mechanisms. Then to explore the role of MAGE-A3 has played in the process of chemotherapy drugs in HCC and lay the foundation for clinical finding ways to improve liver cancer chemotherapy drugs sensitivity.Method1. Human hepatocellsular carcinoma cells Hep G2(wild-type P53) and Huh-7(mutant P53)which expression of MAGE-A3 were cultured in vitro. After treatment with chemotherapy drugs Cisplatin and adriamycin, we detected the cells growth of Hep G2 and Huh-7 cells. By Q-PCR and Western Blot to detect the expression of MAGE-A3 m RNA and protein of this two kinds of cells before and after chemotherapy drugs treatment. 2. si RNA was designed and in-vitro chemosynthesized aiming to MAGE-A3 m RNA, it was transfected into Hep G2 and Huh-7 cells which high expression with MAGE-A3 by using RNAi-Mate transient transfection method. The experimentwere divided idling group(Only plus transfection reagent),negative control group and MAGE-A3 si RNA interference group(interference Group).The transfection efficiency was detected by inverted fluorescence microscope,and the expression of MAGE-A3 m RNA and protein in transfected hepatocellsular carcinoma cells was assessed by Q-PCR and Western Blot. 3. After MAGE-A3 gene silencing of hepatocellsular carcinoma cells, We added different concentration of chemotherapy drugs treatment,by MTT assays to detect the cells growth inhibition of Hep G2 and Huh-7 cells, calculated the cells inhibitory concentration 50(IC50) of chemotherapy drugs in transfected cells,then to compare with cells changement in chemo-sensitivity before and after MAGE-A3 si RNA transfection, and to explore whether the change of chemo-sensitivity was induced by down-regulation of MAGE-A3. 4. Flow cytometry(FCM) assays analysised the cells apoptosis and cell cycle of hepatocellsular carcinoma cells before and after chemotherapy drugs treatment, in order to understand the changement of liver cancer cell apoptosis and cell cycle after MAGE-A3 gene down-regulated. 5. The expression of P53,Bcl-2,Bax,survivin,caspase3 and caspase9 genes m RNAs and proteins were detected by Q-PCR and Western Blot in hepatocellsular carcinoma celllines Hep G2 and Huh-7 after MAGE-A3 silencing and chemotherapy drugs treatment.Results1. MAGE-A3 was expressed in HepG2 and Huh-7 cells, After DDP and ADM treatment, the expression level of MAGE-A3 were increased with the increase of drug dose and action time in Hep G2 and Huh-7 cells. 2. Transfected Hep G2 and Huh-7 cells with MAGE-A3 si RNA 664 fragment for 24 h, 48 h and 72 h, Q-PCR results showed that the expression level of MAGE-A3 were inhibited significantly, and the peak down regulation time was 48 h, the gene silencing efficiency more than 70%. Western Blot detection also confirmed that MAGE-A3 protein expression level decreased significantly when the fragment transfection after 48 h of Hep G2 and Huh-7 cells..3. MTT test results showed that: DDP or ADM applied respectively to Hep G2 and Huh-7 cells for 24 h, 48 h and 72 h, the cells growth were inhibited, all showed dose dependence and time dependence. After transfection MAGE-A3 si RNA, Hep G2 cells sensitivity to DDP and ADM respectively were increased by 1.57 times and 2.15 times, the difference was statistically significant(P<0.05); Also,Huh-7 cells sensitivity to DDP and ADM respectively were increased by 1.22 times and 1.28 times, the difference was statistically significant(P<0.05). 4. Annexin V detection Hep G2 and Huh-7 cells apoptosis, the result shows: When cells cultured without chemotherapy drugs, the apoptosis rate in the interference group of Hep G2 cells were significant increased(P<0.05), while Huh-7 cells in the interference group comparing to the control group, there was no statistically significant(P>0.05).After DDP or ADM treated, the apoptosis rate of Hep G2 and Huh-7 cells in the interference group all were significant increased comparing to the control group(P<0.05), the result remindered us that silencing MAGE-A3 can enhance DDP or ADM cytotoxic drugs effect of Hep G2 and Huh-7cells, and it was more significantly in Hep G2 cells. 5. PI detection Hep G2 and Huh-7 cells cycle, the result shows: When MAGE-A3 silencing, Hep G2 and Huh-7 cells G2 phase proportion were increased,when we added the DDP treatment, comparing to the negative control group,Hep G2 and Huh-7 cells S phase proportion was increased in the interference group, meanwhile, added ADM treatment, comparing to the negative control group,Hep G2 and Huh-7 cells G2 phase proportion was increased in the interference group. 6. Q-PCR and Western Blot to detect apoptosis related factors, and the result shows: after MAGE-A3 silencing added DDP or ADM treatment, in Hep G2 cells, P53, Bax, caspase3 and caspase9 expression increased, and the Bcl-2 and survivin expression decreased, prompted us that the possible mechanisms of MAGE-A3 silencing affect the sensitivity to Hep G2 cells chemotherapy drugs is P53 dependent apoptosis pathway induced; but in Huh-7 cells, mutant P53 expression increased with higher Bax expression and lower survivin expression, while had nothing to do with Bcl-2,caspase3 and caspase9 expression changes, reminder us that the possiblemechanisms of MAGE-A3 silencing affect the sensitivity to Huh-7 cells chemotherapy drugs may involve P53 undependent cell signal transduction pathways.Conclusions1. The expression of MAGE-A3 can be induced by chemotherapy drugs DDP and ADM,MAGE-A3 silencing can enhance the sensitivity of wipe-type P53 and mutant P53 human hepatocellsular carcinoma cells to chemotherapy drugs. 2. Apoptosis involves the process of the MAGE-A3 si RNA promoting chemotherapy drugs to kill hepatocellsular carcinoma cells,it may be more closely relationship with P53 dependent apoptosis pathway. |
PDF Full Text Request