| Aims: Liver cancer is the most5common cancer in china, and the metastasis andrapid growth of hepatocellular carcinoma are the main cause of recurrence and poorprognosis. The invasion and metastasis of tumor cells is a complex and continuous processincluding multiple steps. After invading into the vessel lomen, most cells occur anoikis dueto losing of connection between cells and extracellular matrix. Only a few cells which ownthe ability of anoikis resistance survived and finally formed distant metastasis. Thus,anoikis resistance has been considered as a prerequisite of tumor metastasis. This project isto investigate that AEG-1could promote anoikis resistance by activating PI3K/Aktsignaling in hepatocellular carcinoma cells and effects of autophagy on anoikis resistanceenhanced byAEG-1.Methods: Thirty-six pairs of liver cancer and their adjacent normal liver tissues wereobtained from the patients with histologically confirmed HCC after informed consent wasgiven. The mRNA and protein levels of AEG-1expression in liver cancer and their adjacentnormal liver tissues and four liver cancer cell lines (including SMMC-7721, HepG2,MHCC-97H, and HCC-LM3) were examined by Real-time PCR and Western blot, and therelationship between AEG-1expression and metastasis of liver cancer was assayed. ThepcDNA3.1-AEG-1plasmid which contains the AEG-1CDS sequence was transfected intoSMMC-7721cells to obtain stable cell line with AEG-1over-expression; and psilencer2.0-AEG-1-shRNAs against AEG-1was transfected into HCC-LM3cells to obtainstable cell line with AEG-1knockdown. Cell cycle, proliferation, colony formation andapoptosis were detected by flow cytometry assay, MTT assay, soft agar colony formationassay. The anoikis ratio of AEG-1-overexpressing and-knockdown cells was examied byflow cytometry. Activated caspase-3was detected by Western blot to confirm theoccurrence of anoikis. The activation of PI3K/Akt and ERK1/2pathways was confirmed byprotein detection of key molecule by Western blot. LY294002was used to inhibit theactivation of PI3K/Akt signaling and ERK1/2signaling was block by U0126. Bcl-2andBad, downstream of PI3K/Akt, was examined by Western blot. Autophagy level ofanoikis-resistant HCC cells was confirmed by detection of LC3I/II transform by Westernblot. Atg5siRNA was transfected into cells to down-regulate the autophagy level inAEG-1-overexpressing cells.Results: We detected the expression of AEG-1in thirty-six pairs of humanhepatocellular carcinoma and their corresponding para-carcinoma tissues. In twenty-threepairs of samples, the mRNA and protein levels of AEG-1expression were significantlyhigher (≥2fold) than that in adjacent para-carcinoma tissues. And the expression of AEG-1was closely related to tumor size (p<0.05), differentiation degree (p<0.05), portal invasion(p<0.05) and lymph node metastases (p<0.05). The expression levels of AEG-1mRNA andprotein in HCC cells with high metastasis potential (MHCC-97H, HCC-LM3) were higherthan that in HCC cells with low and moderate metastasis potential (HepG2, SMMC-7721).Compared with SMMC-7721cells with pcDNA3.1empty vector plasmid transfection, themRNA and protein levels of AEG-1expression were significantly enhanced inpcDNA3.1-AEG-1plasmid transfected SMMC-7721cells. Compared with HCC-LM3cellswith psilencer2.0empty vector plasmid transfection, the mRNA and protein levels ofAEG-1expression were significantly reduced in psilencer2.0-AEG-1-shRNAs plasmidtransfected HCC-LM3cells. There were no significant differences in cell cycle, cell proliferation (p>0.05), colony formation (p>0.05) and apoptosis (p>0.05) betweenSMMC-7721-AEG-1cells and SMMC-7721-Vec cells. While, the soft agar colonyformation of SMMC-7721-AEG-1cells was enhanced compared with SMMC-7721-veccells (p<0.05). The cell cycle(p<0.05), proliferation(p<0.05), colony formation(p<0.05),and soft agar colony formation(p<0.05) were significantly inhibited in HCC-LM3-shAEG-1cells compared with HCC-LM3-Vec cells. The apoptosis ratio of AEG-1-knockdownHCC-LM3cells was increased compared to empty plasmid transfected cells (p<0.05). Aftercells were suspension cultured for24h, the anoikis ratio of SMMC-7721-AEG-1cells wassignificantly reduced compared with SMMC-7721-Vec cells, and the anoikis ratio ofHCC-LM3-shAEG-1cells was remarkably increased compared with HCC-LM3-Vec cells.Also, cleaved-caspase-3expression was down-regulated in SMMC-7721-AEG-1cells andup-regulated in HCC-LM3-shAEG-1cells compared with their corresponding controlgroup. p-ERK1/2, p-p85, p-Akt, Bcl-2, and p-Bad protein expressions were increased insuspension cultured SMMC-7721-AEG-1cells compared with vector control group; andthese proteins were reduced in suspension cultured HCC-LM3-shAEG-1cells compared tovector control group. PI3K/Akt inhibitor LY294002could promote the anoikis ratio ofSMMC-7721-AEG-1cells and down-regulate the expression of Bcl-2and p-Bad. ButERK1/2inhibitor U0126can not change the anoikis ratio of SMMC-7721-AEG1cells. Theautophagy levels of anokis-resistant cells were confirmed by detection of autophagy relatedLC3I/II transform. LC3II/LC3I was increased in suspension cultured SMMC-7721cellscompared with normal cultured cells. After suspension cultured for24h, LC3II/LC3Ifurther increased in SMMC-7721-AEG-1cells compared with SMMC-7721-Vec cells.After SMMC-7721-AEG-1cells were transfected with Atg5siRNA, and suspensioncultured for24h, the anoikis ratio was increased compared with non-transfectedSMMC-7721-AEG-1cells (p<0.05).Conclusion: AEG-1enhances the ability of anoikis resistance in hepatocellular carcinoma cells via activating PI3K/Akt signaling and up-regulating Bcl-2andphosphorylated Bad, which involved in the process of liver cancer metastasis; up-regulatedautophagy level may also mediated anoikis resistance induced by AEG-1of HCC cells. |
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