AEG-1Gene Plays A Role In Regulating Multidrug Resistance In Hepatocellular Carcinoma Under Hypoxia Environment

Hepatocellular carcinoma (HCC) is one of the most popular malignancies in the world. However, the therapeutic effect of postoperative chemotherapy was largely diminished by multidrug resistance (MDR) in HCC patients. To explore and reveal the molecular mechanism of MDR is of great importance to improve chemotherapy effect and postoperative survival of HCC patients.Astrocyte elevated gene-1(AEG-1) is a newly discovered oncogene. It is located in human chromosome8(8q22) which is closely related to many malignant tumors. Recent studies have shown that AEG-1plays a key role in the pathogenesis of carcinoma and multidrug resistance. AEG-1is a downstream target molecule of the Ras. It is involved in normal cell transformation, tumor cell proliferation, invasion, metastasis, angiogenesis, tumor pathogenesis and mediates tumor cell autophagy activity and resistance to chemotherapy through the regulation of PI3K/Akt, NF-kB and other signaling pathways. Many studies showed that AEG-1may regulate the expression of multidrug resistance gene1(MDR1) via several signaling pathways, then involved in the regulation of HCC multidrug resistance. However, the mechanism is still unknown.In our study, the expressions of AEG-1、MDR1were firstly detected in HCC tissues, para-cancer tissues and normal liver tissue samples. Further, the expression of AEG-1will be studied under hypoxia environment by RNA interference to investigate the role and molecular mechanism of AEG-1in regulating of MDR1expression in HepG2cell lines.Chapter Ⅰ The expression of AEG-1and MDR1in hepatocellular carcinoma, para-cancer and normal liverObjective To explore the expression difference of AEG-1in hepatocellular carcinoma tissues, para-cancer tissues and normal liver tissues.Methods(1) Immunohistochemical staing was employed to detect distribution and positive expression rate of AEG-1and MDR1protein in30cases of hepatocellular carcinoma tissues, para-cancer tissues and8cases of normal liver tissues (NLs) were regarded as control.(2) Real-time quantitative PCR (qRT-PCR) was respectively employed to detect expression of AEG-1mRNA and MDR1mRNA in30cases of fresh hepatocellular carcinoma tissues, corresponding para-cancer and8cases of NLs were regarded as control. Results(1) Immunohistochemical analysis showed that the distribution of AEG-1and MDR1protein are mainly located in cytoplasm, and the positive expression rate and scores mean of AEG-1and MDR1in hepatocellular carcinoma tissues were both significantly higher than those in para-cancer tissues and NLs (P<0.05). While, there is no significant difference of their positive levels between para-cancer tissues and normal liver tissues.(2) qRT-PCR analysis showed that expression levels of AEG-1mRNA and MDR1mRNA in fresh hepatocellular carcinoma tissues were significantly higher than those in the corresponding para-cancer non tumor tissues and NLs (P<0.05). But there is no significant difference of AEG-1levels between para-cancer tissues and NLs.ConlusionAEG-1and MDR1are highly expressed in hepatocellular carcinoma tissues. But them are lowly expressed in para-cancer tissues and NLs. And there is no significant difference in expression levels of them between para-cancer tissues and NLs. Chapter II The Effect on HepG2cell line Mutiple Resistance via AEG-1Expression under Hypoxia EnvironmentObjective To investigate the effect on HCC mutiple resistance via AEG-1expression under hypoxia environment.Methods(1) HepG2under nomal oxygen environment as control group (group A); add100umol/L CoCl2into HepG2build cell hypoxia model as experimental group (group B); L02under nomal oxygen environment as normal control group (group C).(2)qRT-PCR and Western blotting assay were employed to detect the expression of AEG-1and MDR1in group A、group B、group C at oh、24h、48h(3) MTT assay were employed to detect48h IC50of ADM、5-Fu and DDP in group A、group B at48h.Results(1) qRT-PCR analysis showed that the expression of AEG-1mRNA and MDR1mRNA in group A was markedly higher than those in group C (P<0.01); the expression of AEG-1mRNA and MDR1mRNA in group B was markedly higher than those in group A and group C (P<0.01).(2) Western blotting analysis showed that the expression of AEG-1protein and MDR1protein in group A was markedly higher than those in group C (P<0.01); the expression of AEG-1protein and MDR1protein in group B was markedly higher than those in group A and group C (P<0.01).(3) MTT analysis showed that the48h IC50of ADM、5-Fu and DDP in group B was markedly higher than those in group A, the IC50of ADM was enhanced by2.02fold (P<0.01), and to5-Fu by1.53fold (P<0.05), to DDP by1.90fold (P<0.01).ConlusionAEG-1and MDR1in hepatocellular carcinoma cell lines HepG2with high expression under hypoxia environment. The high expression of AEG-1may mediated multidrug resistance in hepatocellular carcinoma through the up-regulation of MDR1expression. ChapterⅢ AEG-1Gene RNAi Plasmid Vector Construction, Identification and ScreeningObjective To construct AEG-1gene RNAi plasmid vector, screening positive and negative interference plasmid vector.Methods(1)Design target sequence, construct the plasmid vector pGPU6-AEG-1-shRNA. The pGPU6-AEG-1-shRNA-NC is negative interference plasmid vector, pGPU6-AEG-1-shRNA-746, pGPU6-EG-1-hRNA-1490and pGPU6-AEG-1-shRNA-1576are positive interference plasmid vector.(2)Restriction enzymes, detect sequence to identify plasmid vector.(3)Lip2000mediated transfection of HepG2, cultured48hours under hypoxia.(4) Detection of transfection rate. Divided into four groups:group A AEG-1-shRNA-NC; group B AEG-1-shRNA-746; group C AEG-1-shRNA-1490; group D AEG-1-shRNA-1576.(5) Divided into five groups:group A HepG2+CoC12; group B HepG2+CoC12+shRNA-NC; group C HepG2+CoC12+shRNA-746; group D HepG2+CoC12+shRNA-1490; group E HepG2+CoC12+shRNA-1576. qRT-PCR and Western blotting assay were employed to detect the expression of AEG-1in these group.Results(1) Success to construct plasmid vector.(2) Detection of the HepG2cancer cell line transfected plasmid transfection rate, have reached around75%.(3) qRT-PCR, Western blotting analysis showed that the expression of AEG-1mRNA and protein in group C lower than other groups (P<0.01), and the expression of AEG-1mRNA and protein in group B and group A have no obvious difference. ConlusionSuccess to construct pGPU6-AEG-1-shRNA plasmid vetor. On target gene AEG-1interference effect better is the pGPU6-AEG-1-ShRNA-746plasmid vector; pGPU6-AEG-1-shRNA-NC on HepG2expression of AEG-1in cells and have little effect. Chapter Ⅳ Target to AEG-1expression effect on HepG2cell line Mutiple Resistance by RNA interference under Hypoxia EnvironmentObjective To investigate the effect on HepG2mutiple resistance via AEG-1expression under hypoxia environment by RNA interference.Methods(1) positive interference plasmid vector pGPU6AEG-1RNAi+and negative interference plasmid vector pGPU6AEG-1RNAi-transfected into HepG2cells lines.(2) Divided into three groups:group A HepG2+CoCl2; group B HepG2AEG-1RNAi-+CoCl2; group C HepG2AEG-1RNAi+CoCl2.(2)qRT-PCR assay was employed to detect the expression of AEG-1and MDR1mRNA in group A、group B、group C at oh、24h、48h; Western blotting assay was employed to detect the expression of AEG-1 and MDR1protein in group A、group B、group C at oh、24h、48h.(3) MTT assay were employed to detect48h IC50of ADM、5-Fu and DDP in group A. group B at48h.Results(1) qRT-PCR analysis showed that the expression of AEG-1mRNA and MDR1mRNA in group C was markedly lower than those in group A (P<0.01).(2) Western blotting analysis showed that the expression of AEG-1protein and MDR1protein in group C was markedly lower than those in group A(P<0.01).(3) MTT analysis showed that the48h IC50of ADM.5-Fu and DDP in group C was markedly lower than those in group A, the IC50of ADM was enhanced by0.59fold (P<0.01), and to5-Fu by0.66fold (P<0.05), to DDP by0.69fold (P<0.05).Conlusion(1)Specific inhibition of the expression of AEG-1in HepG2cell lines under hypoxic microenvironment, can significantly down-regulate the expression of MDR1, suggesting that AEG-1as an upstream genes of MDR1, can up-regulation the expression of MDR1.(2) Specific inhibition of the expression of AEG-1in HepG2cell lines under hypoxic microenvironment, the48hours IC50of ADM,5-Fu, DDP in HepG2cells lines were reduced, prompting inhibits the expression of AEG-1can reversal of HepG2cells lines multidrug resistance.