The E3 ubiquitin ligase Fbxw7 functions as a general tumor suppressor by targeting several well-known oncoproteins for ubiquitination and proteasomal degradation. However, the clinical significance of Fbxw7 and the mechanisms involved in the anti-cancer effect of Fbxw7 in HCC are not clear. The Fbxw7 and YAP expression in 60 samples of surgical resected HCC and matched normal tumor-adjacent tissues were assessed using IHC or immunoblotting. Flow cytometry, caspase 3/7 activity assay, BrdU cell proliferation assay and MTT assay were used to detect proliferation and apoptosis of HCC cells. The regulatory effect of Fbxw7 on YAP in HCC cells was confirmed by qRT-PCR, immunoblotting and immunofluorescence. Co-immunoprecipitation was used to analyze interaction between YAP and Fbxw7. Nude mice subcutaneous injection, Ki-67 staining and TUNEL assay were used to evaluate tumor growth and apoptosis in vivo. In this study, we found that Fbxw7 expression was impaired in HCC tissues and loss of Fbxw7 expression was correlated with poor clinicopathological features including large tumor size, venous infiltration, high pathological grading and advanced TNM stage. Additionally, we demonstrated that patients with positive Fbxw7 expression had a better 5-year survival and Fbxw7 was an independent factor for predicting the prognosis of HCC patients. We confirmed that Fbxw7 inhibited HCC by inducing both apoptosis and growth arrest. Elevated YAP expression was observed in the same cohort of HCC tissues. Pearson's correlation coefficient analysis indicated that Fbxw7 was inversely associated with YAP protein expression in HCC tissues. We also found that Fbxw7 regulated YAP protein abundance by targeting YAP for ubiquitination and proteasomal degradation in HCC. Furthermore, restoring YAP expression partially abrogated Fbxw7 induced HCC cell apoptosis and growth arrest in vitro and in vivo. These results indicate that Fbxw7 may serve as a prognostic marker and that YAP may be a potential target of Fbxw7 in HCC.

MircroRNA-130b (miR-130b) is proposed as a novel tumor-related miRNA and has been found to be significantly dysregulated in tumors. In this study, the expression level of miR-130b was found to be obviously higher in hepatocellular carcinoma (HCC) tissues than that in nontumor tissues. Further, miR-130b was expressed at significantly higher levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that high-expression of miR-130b was prominently correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage in HCC. Elevated miR-130b expression was observed in all HCC cell lines (HepG2, SMMC-7721, Huh7, Hep3B and MHCC97H) as compared with that in a nontransformed hepatic cell line (LO2). Furthermore, an inverse correlation between miR-130b and E-cadherin and a positive correlation between miR-130b and Vimentin were observed in HCC tissues. Down-regulation of miR-130b expression reduced invasion and migration in both Hep3B and MHCC97H cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) was inversely correlated with miR-130b expression in HCC tissues. In addition, down-regulation of miR-130b restored PPAR-γ expression and subsequently suppressed epithelial-mesenchymal transition (EMT) in HCC cells. We identified PPARγ as a direct target of miR-130b in HCC in vitro. Notably, PPAR-γ knockdown abolished down-regulation of miR-130b-inhibited EMT in MHCC97H cells. In conclusion, miR-130b may promote HCC cell migration and invasion by inhibiting PPAR-γ and subsequently inducing EMT.

Background: Fibulin-5 has been considered as a tumor suppressor through inhibiting tumor growth and invasion. Reduced expression of Fibulin-5 is frequently observed in various human cancers. In this study, we investigate the clinical significance of Fibulin-5 and its role in hepatocellular carcinoma (HCC) cell migration and invasion. Methods: The expression of Fibulin-5 was evaluated by qRT-PCR and immunoblotting in HCC and matched noncancerous tissues. Fibulin-5 was over-expressed or knocked down by a retrovirus-mediated expression plasmid or a specific siRNA in HCC cells. Boyden chamber and Transwell assays were used to test HCC cell migration and invasion. Immunostaining was performed to determine matrix metalloproteinase-7 (MMP-7) expression in HCC specimens. MMP-7 retroviruses and siRNA were used to alter MMP-7 expression in HCC cells. Results: In our study, the expression levels of Fibulin-5 protein and mRNA were down-regulated in HCC tissues as compared with those in matched noncancerous tissues. Reduced expression of Fibulin-5 was observed in all HCC cell lines (HepG2, SMMC-7721, MHCC97L, Hep3B, MHCC97H and HCC-LM3) as compare with that in a non-transformed hepatic cell line (LO2). Low expression of Fibulin-5 was significantly correlated with poor prognostic features including multiple tumor nodes, venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage. Furthermore, we demonstrated that Fibulin-5 was a novel independent prognostic marker for predicting 5-year survival of HCC patients. Our in vitro studies showed that Fibulin-5 overexpression inhibited HCC cell migration and invasion. While Fibulin-5 knockdown increased the number of migrated and invaded HCC cells. Fibulin-5 negatively regulated MMP-7 abundance in HCC cells. Moreover, the inverse correlation between Fibulin-5 and MMP-7 expressions was observed in HCC tissues. Mechanistically, we disclosed that MMP-7 knockdown reduced the number of migrated and invaded HCC cells. Restoring MMP-7 expression abrogated the suppressive effect of Fibulin-5 on HCC cell migration and invasion in vitro , suggesting that Fibulin-5 exerted its anti-metastatic function, at least in part, by down-regulating the expression of MMP-7 in HCC cells. Conclusions: These results indicate that Fibulin-5 may serve as a prognostic biomarker and inhibits HCC invasion and metastasis by suppressing MMP-7 expression.

Background: The deregulation of E-cadherin has been considered as a leading cause of hepatocellular carcinoma (HCC) metastasis. BCL6 corepressor-like 1 (BCORL1) is a transcriptional corepressor and contributes to the repression of E-cadherin. However, the clinical significance of BCORL1 and its role in the metastasis of HCC remain unknown. Methods: Differentially expressed BCORL1 between HCC and matched tumor-adjacent tissues, HCC cell lines and normal hepatic cell line were detected by Western blot. The expression of BCORL1 was altered by siRNAs or lentivirus-mediated vectors. Transwell assays were performed to determine HCC cell invasion and migration. Results: Increased expression of BCORL1 protein was detected in HCC specimens and cell lines. Clinical association analysis showed that BCORL1 protein was expressed at significant higher levels in HCC patients with multiple tumor nodes, venous infiltration and advanced TNM tumor stage. Survival analysis indicated that high expression of BCORL1 protein conferred shorter overall survival (OS) and recurrence-free survival (RFS) of HCC patients. Multivariate Cox regression analysis disclosed that BCORL1 expression was an independent prognostic marker for predicting survival of HCC patients. Our in vitro studies demonstrated that BCORL1 prominently promoted HCC cell migration and invasion. Otherwise, an inverse correlation between BCORL1 and E-cadherin expression was observed in HCC tissues. BCORL1 inversely regulated E-cadherin abundance and subsequently facilitated epithelial-mesenchymal transition (EMT) in HCC cells. Notably, the effect of BCORL1 knockdown on HCC cells was abrogated by E-cadherin silencing. Conclusions: BCORL1 may be a novel prognostic factor and promotes cell migration and invasion through E-cadherin repression-induced EMT in HCC.