KDM5A silencing transcriptionally suppresses the FXYD3-PI3K/AKT axis to inhibit angiogenesis in hepatocellular cancer via miR-433 up-regulation.

Hepatocellular cancer (HCC) has been reported to belong to one of the highly vascularized solid tumours accompanied with angiogenesis of human umbilical vein endothelial cells (HUVECs). KDM5A, an attractive drug target, plays a critical role in diverse physiological processes. Thus, this study aims to investigate its role in angiogenesis and underlying mechanisms in HCC. ChIP-qPCR was utilized to validate enrichment of H3K4me3 and KDM5A on the promotor region of miR-433, while dual luciferase assay was carried out to confirm the targeting relationship between miR-433 and FXYD3. Scratch assay, transwell assay, Edu assay, pseudo-tube formation assay and mice with xenografted tumours were conducted to investigate the physiological function of KDM5A-miR-433-FXYD3-PI3K-AKT axis in the progression of HCC after loss- and gain-function assays. KDM5A p-p85 and p-AKT were highly expressed but miR-433 was down-regulated in HCC tissues and cell lines. Depletion of KDM5A led to reduced migrative, invasive and proliferative capacities in HCC cells, including growth and a lowered HUVEC angiogenic capacity in vitro. Furthermore, KDM5A suppressed the expression of miR-433 by demethylating H3K4me3 on its promoterregion. miR-433 negatively targeted FXYD3. Depleting miR-433 or re-expressing FXYD3 restores the reduced migrative, invasive and proliferative capacities, and lowers the HUVEC angiogenic capacity caused by silencing KDM5A. Therefore, KDM5A silencing significantly suppresses HCC tumorigenesis in vivo, accompanied with down-regulated miR-433 and up-regulated FXYD3-PI3K-AKT axis in tumour tissues. Lastly, KDM5A activates the FXYD3-PI3K-AKT axis to enhance angiogenesis in HCC by suppressing miR-433.

The prognostic value of Niemann-Pick C1-like protein 1 and Niemann-Pick disease type C2 in hepatocellular carcinoma.

Niemann-Pick C1-like 1 (NPC1L1) and Niemann-Pick C2 (NPC2) is a critical mediator of cholesterol absorption. The aim of the present study was to investigate the prognostic value of NPC1L1 and NPC2 in human primary hepatocellular carcinoma (HCC). The expression level of NPC1L1 and NPC2 were evaluated by Immunohistochemistry, Westen blot and Real-time Quantitative PCR. Protein expression level in tissue was represented by integral optic density (IOD). For prognosis analyses, outcome-based cut-point was calculated by X-tile software. Kaplan-Meier analysis, Cox regression analysis were used evaluate prognostic value of NPC1L1 and NPC2 and NPC1L1/NPC2 combination. Both of NPC1L1 and NPC2 were significantly decreased in HCC tissues than peritumoral liver tissues (61 pairs of tissue for Immunohistochemistry and 10 pairs of tissues for Western blot and Real-time Quantitative PCR), respectively. (n=61: p=0.0005 for NPC1L1 and p=0.0001 for NPC2; n=10: p=0.0002 for NPC1L1 and p=0.0489 for NPC2). Kaplan-Meier analyses in 265 HCC cases were showed that the low expression level of NPC1L1 and NPC2 and NPC1L1/NPC2 combination were significantly correlated with poor overall survival (OS) and shorter time to recurrence (TTR). In addition, univariate and multivariate Cox analyses showed that the expression level of NPC1L1/NPC2 combination in HCC was an independent prognostic factor for OS and TTR. Conclusion: NPC1L1 and NPC2 were lowly expressed in HCC compared with peritumoral liver tissues, and low expression of NPC1L1 and NPC2 in HCC tissues may indicate poor outcome of HCC patients after surgery. NPC1L1/NPC2 combination is an independent prognostic factor for OS and TTR in postoperative HCC patients.

Association of tumor-associated fibroblasts with progression of hepatocellular carcinoma.

Interaction between tumor and stromal cells plays an important role in cancer progression. The aim of this study was to explore the effects of tumor-associated fibroblasts on regulation of hepatocellular carcinoma (HCC) progression. Sixty-five cases of HCC and the corresponding normal liver tissues were recruited for immunohistochemical assessment of α-smooth muscle actin (α-SMA) expression, a biomarker for activated fibroblasts. Clinicopathological data were also collected from HCC patients for association with α-SMA expression. Primary cell culture of fibroblasts from HCC tissues was used to generate conditioned medium for testing the effect on regulation of HCC cell migration capacity in the transwell cell migration assay. α-SMA protein was expressed in 84.0 % (21 out 25 cases) of the fibroblasts from the metastatic HCCs, 45 % (18/40) from HCCs without metastasis, and 19.2 % (5/26) from normal liver tissues, difference of which was statistically significant (P < 0.01). The expression of α-SMA protein in HCC tissues was associated with tumor thrombosis, poor pathology grade, advanced clinical stages, and lymph node metastasis. The conditioned medium from the primary cultured fibroblasts with α-SMA expression significantly promoted the migration capacity of HCC Hep3B cells compared to the heat-inactivated conditioned medium. The data from the current study demonstrated that expression of α-SMA protein in HCC fibroblasts associated with tumor metastasis and advanced clinical stages and that the conditioned medium from α-SMA-positive fibroblasts enhanced HCC cell migration. This study indicates that α-SMA protein might serve as a biomarker to predict HCC progression.

Overexpression of aspartyl-(asparaginyl)-beta-hydroxylase in hepatocellular carcinoma is associated with worse surgical outcome.

UNLABELLED: The association between the overexpression of aspartyl-(asparaginyl)-beta-hydroxylase (AAH) and the invasiveness of hepatocellular carcinoma (HCC) in vitro has been reported. However, the prognostic value of AAH expression in HCC remains unclear. The purpose of this study was to investigate the relationship between AAH expression, tumor recurrence, and patient survival. We identified AAH as the most overexpressed gene in HCC by way of complementary DNA microarray hybridization. A prospective study of 233 patients undergoing curative resection indicated that AAH expression was an independent factor affecting recurrence (hazard ratio [HR] 3.161, 95% confidence interval [CI] 2.115-4.724, P < 0.001) and survival (HR 2.712, 95% CI 1.734-4.241, P < 0.001). Patients with AAH overexpression had a poorer prognosis than those with AAH underexpression (P < 0.001 for both recurrence and survival). In Barcelona Clinic Liver Cancer stage A patients with AAH overexpression or underexpression, the tumor recurrence and survival rates were also statistically different (45% and 85% versus16% and 33% in 1- and 3-year cumulative recurrence rates, respectively; 73% and 37% versus 90% and 80% in 1- and 3-year survival rates, respectively; P < 0.001 for both). Furthermore, in stage A patients with tumors measuring < or =5 cm in diameter, the time to recurrence was 26.7 +/- 1.6 versus 51.9 +/- 2.8 months, and the 1- and 3- year survival rates were 97% and 52% versus 100% and 90% in AAH overexpression and underexpression patients, respectively (P < 0.001 for both). CONCLUSION: AAH overexpression in HCC is strongly correlated with worse surgical outcome, and this molecule likely provides a more precise prognostic predictor in early stage HCCs.