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Objective To explore the effect of myotubularin-related protein 6(MTMR6)on the invasion of hepatocellular carcinoma cell line HepG2 and the potential molecular mechanism.Methods By analyzing the sequencing results of liver cancer tissues and adjacent tissues in Gene Expression Omnibus(GEO)database,MTMR6 gene was screened out,and Spearman analysis was used to analyze the correlation of MTMR6 and pathway in the Cancer Genome Atlas(TCGA)database.Finally,the interaction between MTMR6 and signaling pathway proteins was analyzed with Genemania database.Then the expression of MTMR6 in human normal liver cell line LO-2 and hepatoma cell lines Huh-7 and HepG2 were measured and compared among the cell lines.Then HepG2 cells was selected as the study object.After MTMR6 gene was knocked down or over-expressed in HepG2 cells,Transwell assay was employed to observe invasion ability,and Western blotting was adopted to detect the expression of MTMR6,PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR MMP-2 and MMP-9.Results The expression of MTMR6 was significantly higher in the hepatocellular carcinoma tissues than the paracancer tissues,and it was in a positive linear correlation with PI3K/AKT/mTOR signaling pathway(P＜0.01),showing interaction with PI3K,AKT and mTOR.The expression level of MTMR6 was significantly higher in the HepG2 cells than the LO-2 and Huh-7 cells(P＜0.01).Over-expression of MTMR6 obviously enhanced invasion ability(P＜0.01),while its knockdown decreased the ability(P＜0.01)in HepG2 cells.Knockdown of MTMR6 gene also resulted in decreased phosphorylation of PI3K,AKT and mTOR,and expression levels of MMP-2 and MMP-9(P＜0.01),while over-expression of MTMR6 promoted the phosphorylation of PI3K,AKT and mTOR,and up-regulated the expression of MMP-2 and MMP-9(P＜0.01).In addition,LY294002(a specific PI3K inhibitor)treatment could block the PI3K/AKT/mTOR pathway and down-regulate the expression of MMP-2 and MMP-9(P＜0.01),but had no effect on MTMR6 expression.Conclusion MTMR6 may promote the invasion of hepatoma cells through activation of PI3K/AKT/mTOR signaling pathway.
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Objective:To investigate the effect of rapamycin on hepatic ischemia-reperfusion injury (HIRI) in Sprague Dawley (SD) rats and its underlying mechanism.Methods:Forty-eight specific pathogen-free SD male rats with the body weight of 180-200 g and the age of 4-8 weeks were randomly divided into 3 groups, 16 rats each group. In the rapamycin group, the rats were injected with rapamycin intraperitoneally everyday lasting for 3 days before the surgery, and in the model group and the sham group, the rats were injected with normal saline intraperitoneally. The HIRI model was performed in the rapamycin group and the model group. Serum of 8 rats was randomly harvested from each group at 2 h and 24 h after the surgery and was used to detect level of alanine aminotransferase(ALT), total bilirubin, and lactate dehydrogenase. At the meantime, liver tissues were collected for HE staining, and enzyme-linked immunosorbent assay of superoxide dismutase(SOD), glutathione, hexokinase 2, phosphofructokinase 1(PFK1), and adenosine triphosphate. Polymerase chain reaction and Western blots were used to determine the levels of mammalian target of rapamycin(mTOR), ribosomal protein S6 kinase 1(S6K1), and protein kinase B and their phosphorylation levels respectively.Results:Two hours post the surgery, the serum level of ALT(150.9±18.7) U/L, total bilirubin(5.15±0.69) μmol/L, and lactate dehydrogenase(9 547±365) U/L were higher in the model group than sham group (42.4±10.7) U/L, (2.48±0.24) μmol/L, (4 424±376) U/L and rapamycin group (87.7±11.2) U/L, (3.09±0.12) μmol/L, (8 268±264) U/L, and all differences were statistically significant (all P<0.05). HE staining and serum assay showed that the lesion of liver tissuesand of liver function were damaged in the model group, and mitigated in the rapamycin group at 2h and 24h after the surgery. At 2h and 24h after the surgery, liver SOD, glutathione, hexokinase 2, PFK1, and adenosine triphosphate in the model group were lower than those in the sham group and the rapamycin group, and all differences were statistically significant (all P<0.05). The relative levels of mTOR, S6K1, and their phosphorylation level in the model group were higher than those in the sham group and the rapamycin group at 2 h and 24 h after the surgery, but the relative levels of protein kinase B and phosphorylated protein kinase B were lower than those in the sham group and the rapamycin group, and all differences were statistically significant (all P<0.05). Conclusions:Rapamycin improves glucose metabolism and reduces oxidative stress via upregulating the phosphorylated protein kinase B through inhibition of mTOR signaling pathway, thus alleviates HIRI in rats.