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Objective:To investigate the therapeutic effect and potential molecular mechanisms of cyclin-dependent kinase inhibitor-73 (CDKI-73), the Rab11 inhibitor, on liver fibrosis.Methods:Human LX2 cells were divided into four groups: negative control group, transforming growth factor-β (TGF-β) group, CDKI-73 group and TGF-β+ CDKI-73 group. Fifteen 5-week-old female C57 mice with body weight of (18.04±0.62) g were divided into 3 groups with 5 mice in each group: control group (intraperitoneal injection of olive oil + vehicle gavage), carbon tetrachloride (CCl 4) group (intraperitoneal injection of CCl 4 + vehicle gavage) and CCl 4+ CDKI-73 group (intraperitoneal injection of CCl 4+ CDKI-73 gavage). Another 15 5-week-old female C57 mice with body weight of (18.06±0.34) g were divided into 3 groups with 5 mice in each group: sham operation group (Sham), bile duct ligation (BDL) group + vehicle group (BDL+ vehicle gavage) and bile duct ligation+ CDKI-73 group (BDL+ CDKI-73 gavage). The expression of α-smooth muscle actin (α-SMA) and fibronectin(FN)in LX2 cells were analyzed by Western blot. Masson and Sirius red were used to examine the liver fibrosis after CDKI-73 treatment in vivo. Immunohistochemistry (IHC) was utilized to examine the expression of α-SMA in mice liver. Results:Collagen content assessed by Sirius red and Masson staining and α-SMA expression evaluated by IHC were all increased in CCl 4 group compared with control group ( q=38.47, 24.99, 36.79). Moreover, the collagen content and α-SMA expression in CCl 4 + CDKI-73 treatment group were obviously decreased compared with CCl 4 group ( q=24.72, 14.87, 27.50), and the differences were statistically significant (all P<0.001). Compared with Sham group, collagen content and α-SMA expression in bile duct ligation group were increased ( q=28.23, 41.01, 44.16). Furthermore, in BDL group, after treatment with CDKI-73, the collagen content and α-SMA expression were notably decreased ( q=22.88, 34.31 and 33.97, all P<0.001). Consistent with in vivo results, the relative expression levels of α-SMA and FN protein in TGF-β group were higher than those in TGF-β+ CDKI-73 group (α-SMA: 3.71±0.34 vs. 1.28±0.31; FN: 3.21±0.39 vs. 0.83±0.06, all P<0.001). The mRNA relative expression levels of α-SMA and FN in TGF-β group were higher than those in TGF-β+ CDKI-73 group, and the differences were statistically significant ( P<0.001). However, the relative expression of TGF-β receptor Ⅱ protein in CDKI-73 group was higher than those in negative control group (4.68±0.63 vs. 1.00±0.22, P=0.004). The relative expression level of phosphorylated SMAD2 in TGF-β+ CDKI-73 group was lower than those in TGF-β group (1.67±0.24 vs. 3.99±0.44, P<0.001). Transwell assay showed that 0.5 μmol/L CDKI-73 could effectively inhibit the migration of LX2 cells, and the inhibitory ability became stronger with the increase of CDKI-73 concentration. Conclusion:CDKI-73 can inhibit the activation of hepatic stellate cells and liver fibrosis by inhibiting Rab11-dependent TGF-β signaling pathway both in vivo and in vitro.
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【Objective】 To investigate the cell death-inducing effect of methyl rosmarinate (MR) on human hepatoma Hep-3B and SK-Hep1 cells and their potential mechanisms. 【Methods】 The effects of MR on the viability of Hep-3B, SK-Hep1 and MIHA cells were determined by cell counting kit-8 (CCK-8) assay. The morphological changes of three kinds of cells treated with different concentrations of MR were observed by optical microscopy. EdU assay and flow cytometry were used to detect the proliferation and apoptosis of Hep-3B and SK-Hep1 cells. Transwell assay was used to study the effects of MR on the migration and invasion of Hep-3B and SK-Hep1 cells. Western blotting was used to evaluate the protein expression levels of apoptosis, EMT and Akt/mTOR signaling pathways. 【Results】 After treated with different concentrations of MR (0~200 μmol/L) for 48 h, Hep-3B and SK-Hep1 cells activities were significantly decreased in a concentration-dependent manner (P<0.01), while there was no significant effect on MIHA cell activity (P>0.05), and the IC50 of Hep-3B and SK-Hep1 cells were 102.5 and 99.3 μmol/L, respectively. MR treatment (0-150 μmol/L) significantly inhibited the proliferation of Hep-3B and SK-Hep1 cells (P<0.05), while cell detachment and shrinkage were observed by optical microscopy on the Hep-3B and SK-Hep1 cells, while the morphology of MIHA cells was not changed. Compared with the control group, MR (100, 150 μmol/L) induced apoptosis in Hep-3B and SK-Hep1 cells, the expression levels of the pro-apoptotic proteins Bax and cleaved PARP were significantly increased (P<0.05), while the expression level of the anti-apoptotic protein Bcl-2 was significantly decreased (P<0.05). MR (100, 150 μmol/L) also inhibited the migration and invasion of HCC cells, significantly increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin compared with the control group (P<0.05). Finally, Western blotting results showed that the expressions of p-Akt and p-mTOR in Hep-3B and SK-Hep1 treated by MR were significantly reduced in a dose-dependent manner, suggesting that MR may play an anti-cancer role by inhibiting Akt/mTOR signaling pathway. 【Conclusion】 MR can promote apoptosis in Hep-3B and SK-Hep1 cells, which may be closely related to the inhibition of Akt/mTOR signaling pathway.
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Objective To investigate the clinical roles of lysine methyltransferase 5A(KMT5A)in human hepatocellular carcinoma(HCC)and its functions in cell migration and invasion. Methods The expression levels of KMT5A of 60 cases were detected by immunohistochemistry(IHC). KMT5A siRNA was used to down-regulate the expression of KMT5A in SMMC-7721 cells. Cell migration and invasion were measured by wound healing assays and transwell assays,respectively. Immunoblotting was used to detect the expression of MMP-2 after siRNA trans-fection. miR-186 mimics were transfected into SMMC-7721 cells and the mRNA levels of KMT5A was detected by qRT-PCR after transfection. Results High expression of KMT5A was associated with large tumor diameter (>5 cm,P=0.047)and advanced TNM stage(Ⅲ+Ⅳ,P=0.035). The expression of KMT5A was knocked down by siRNA in SMMC-7721 cells. Down-regulation of KMT5A suppressed cell migration(P=0.031,P=0.006)and invasion(P=0.010),and impaired MMP-2 expression(P=0.040). Overexpression of miR-186 could significantly inhibit the expression of KMT5A(P = 0.007). Conclusions Over-expression of KMT5A in HCC tissues associ-ates with poor clinical features. KMT5A knockdown inhibits the migration and invasion on HCC cells.
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<p><b>OBJECTIVE</b>To investigate the expression of microRNA-218 (miR-218) and its role in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Forty-six pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-218 expression using qRT-PCR. A miR-218 mimic was transfected into HepG2 cells, and the cell viability and apoptosis were analyzed by MTT assay and flow cytometry, and the potential targets of miR-218 were detected by qRT-PCR and Western blotting.</p><p><b>RESULTS</b>The expressions of miR-218 in HCC tissues were significantly down-regulated compared to those in the adjacent tissues (P<0.05). Down-regulation of miR-218 was found to correlate significantly with the tumor size (>5 cm) and an advanced TNM stage (III+IV) (P<0.05). Ectopic expression of miR-218 in HepG2 cells resulted in suppressed cell proliferation and enhanced cell apoptosis as well as the down-regulation of Bmi-1 and CDK6 mRNA and protein expressions (P<0.05).</p><p><b>CONCLUSION</b>The low-expression of miR-218 is correlated with malignant clinicopathological characteristics of HCC, and miR-218 may inhibit cell proliferation and promote cell apoptosis by down-regulating Bmi-1 and CDK6 in HCC.</p>