RNF125­mediated ubiquitination of MCM6 regulates the proliferation of human liver hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortality worldwide. Minichromosome maintenance proteins (MCMs), particularly MCM2-7, are upregulated in various cancers, including HCC. The aim of the present study was to investigate the role of MCM2-7 in human liver HCC (LIHC) and the regulation of the protein homeostasis of MCM6 by a specific E3 ligase. Bioinformatics analyses demonstrated that MCM2-7 were highly expressed in LIHC compared with corresponding normal tissues at the mRNA and protein levels, and patients with LIHC and high mRNA expression levels of MCM2, MCM3, MCM6 and MCM7 had poor overall survival rates. Cell Counting Kit-8 and colony formation assays revealed that the knockdown of MCM2, MCM3, MCM6 or MCM7 in Huh7 and Hep3B HCC cells inhibited cell proliferation and colony formation. In addition, pull-down, co-immunoprecipitation and ubiquitination assays demonstrated that RNF125 interacts with MCM6 and mediates its ubiquitination. Furthermore, co-transfection experiments indicated that RNF125 promoted the proliferation of HCC cells mainly through MCM6. In summary, the present study suggests that the RNF125-MCM6 axis plays an important role in the regulation of HCC cell proliferation and is a promising therapeutic target for the treatment of LIHC.

Genomic and transcriptomic profiling reveals key molecules in metastatic potentials and organ-tropisms of hepatocellular carcinoma.

Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2.

Purpose: Smurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC. Methods: The expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial-mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model. Results: Smurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway. Conclusion: The expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.

S100A6 promotes proliferation and migration of HepG2 cells via increased ubiquitin-dependent degradation of p53.

PURPOSE: S100A6 protein (calcyclin), a small calcium-binding protein of the S100 family, is often upregulated in various types of cancers, including hepatocellular carcinoma (HCC). The aim of this study was to illustrate the molecular mechanism of S100A6 in regulating the proliferation and migration of HCC cells. METHODS: The expressions of S100A6 in human HCC and adjacent non-tumor liver specimens were detected using immunoblotting and quantitative PCR (qPCR). The recombinant glutathione S-transferase (GST)-tagged human S100A6 protein was purified and identified. After treatment with S100A6, the proliferation of HepG2 cells was detected by the MTT and colony formation assay, and the migration of HepG2 cells was investigated by the transwell migration assay; the protein levels of cyclin D1 (CCND1), E-cadherin, and vimentin were also tested by immunoblotting. The effect of S100A6 on p21 and nuclear factor-κB pathway was verified by performing the dual luciferase assay. Then, the expression of p21 and its transcription activator, p53, was examined using immunoblotting and qPCR, the ubiquitination of which was investigated through co-immunoprecipitation. RESULTS: It was found that the level of S100A6 was higher in the HCC tissues than in the adjacent non-tumor liver specimens. Exogenous overexpression of S100A6 promoted the proliferation and migration of HepG2 cells. S100A6 was observed to regulate p21 mRNA and protein expression levels and decrease p53 protein expression level, not mRNA level, by promoting the ubiquitination of p53 via the proteasome-dependent degradation pathway. CONCLUSION: Our study indicated that S100A6 overexpression could promote the proliferation and migration of HCC cells by enhancing p53 ubiquitin-dependent proteasome degradation, ultimately regulating the p21 expression level.

Proprotein Convertase Subtilisin/Kexin Type 9 Promotes Gastric Cancer Metastasis and Suppresses Apoptosis by Facilitating MAPK Signaling Pathway Through HSP70 Up-Regulation.

OBJECTIVE: To examine the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on gastric cancer (GC) progression and prognosis, and to explore the underlying mechanism. METHODS: PCSK9 expression levels in human GC tissues were determined by quantitative real-time PCR, western blotting, and immunohistochemical assay. PCSK9 serum levels were detected by enzyme-linked immunosorbent assay. The relationships of PCSK9 and GC progression and survival were analyzed using the Chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. The effect of PCSK9 on cell invasion, migration, and apoptosis were determined in human GC cell lines and mouse xenograft model separately using PCSK9 knockdown and overexpression strategies. The PCSK9 interacting molecules, screened by co-immunoprecipitation combined with LC-MS/MS, were identified by immunofluorescence localization and western blotting. Additionally, the mitogen-activated protein kinase (MAPK) pathway was assessed by western blotting. RESULTS: PCSK9 mRNA and protein levels were significantly elevated in GC tissues compared with the paired normal tissues at our medical center (P < 0.001). Notably, the up-regulation of PCSK9 expression in GC tissues was related to tumor progression and poor survival. GC patients had higher serum levels of PCSK9 than the age-matched healthy controls (P < 0.001); PCSK9 promoted invasive and migratory ability and inhibited apoptosis in GC cells with no apparent affection in cell proliferation. The silencing of PCSK9 reversed these effects, suppressing tumor metastasis in vitro and in vivo. Furthermore, PCSK9 maintained these functions through up-regulating heat shock protein 70 (HSP70), ultimately facilitating the mitogen-activated protein kinase (MAPK) pathway. CONCLUSION: Collectively, our data revealed that high PCSK9 expression levels in GC tissue were correlated with GC progression and poor prognosis and that PCSK9 could promote GC metastasis and suppress apoptosis by facilitating MAPK signaling pathway through HSP70 up-regulation. PCSK9 may represent a novel potential therapeutic target in GC.

MiR-146a attenuates liver fibrosis by inhibiting transforming growth factor-ß1 mediated epithelial-mesenchymal transition in hepatocytes.

Epithelial-mesenchymal transition (EMT) has emerged as a vital process in embryogenesis, carcinogenesis, and tissue fibrosis. Transforming growth factor-beta 1 (TGF-ß1)-mediated signaling pathways play important roles in the EMT process. MicroRNA-146a (miR-146a) has been suggested as a significant regulatory molecule in fibrogenesis. Therefore, the present study aimed to evaluate the effect of miR-146a on the EMT of hepatocytes and to investigate the role of overexpressing miR-146a on rat hepatic fibrosis. The results showed that the miR-146a level decreased during the EMT process of L02 hepatocytes induced by TGF-ß1 in vitro. Moreover, miR-146a overexpression led to significant reduction of EMT-related markers expression in hepatocytes. Subsequent experiments revealed that miR-146a attenuated the EMT process in hepatocytes by targeting small mothers against decapentaplegic (SMAD) 4. Meanwhile, restoration of SMAD4 expression rescued the inhibitory effect of miRNA-146a on EMT. Further in vivo studies revealed that intravenous injection of miR-146a-expressing adenovirus (Ad-miR-146a) successfully restored the miR-146a levels and mitigated fibrogenesis in the livers of CCl4-treated rats. More importantly, after Ad-miR-146a treatment, inhibition of both EMT traits and SMAD4 expression was observed. The results of the present study showed that miR-146a/SMAD4 is a key signaling cascade that inhibits hepatocyte EMT, and the introduction of miR-146a might present a promising therapeutic option for liver fibrosis.

Optimizing methods for the study of intravascular lipid metabolism in zebrafish.

The zebrafish (Danio rerio) is a useful vertebrate model for use in cardiovascular drug discovery. The present study aimed to construct optimized methods for the study of intravascular lipid metabolism of zebrafish. The lipophilic dye, Oil Red O, was used to stain fasting zebrafish one to eight days post-fertilization (dpf) and to stain 7-dpf zebrafish incubated in a breeding system containing 0.1% egg yolk as a high-fat diet (HFD) for 48 h. Three-dpf zebrafish were kept in CholEsteryl boron-dipyrromethene (BODIPY) 542/563 C11 water for 24 h which indicated the efficiency of CholEsteryl BODIPY 542/563 C11 intravascular cholesterol staining. Subsequently, 7-dpf zebrafish were incubated in water containing the fluorescent probe CholEsteryl BODIPY 542/563 C11 and fed a high-cholesterol diet (HCD) for 10 d. Two groups of 7-dpf zebrafish were incubated in regular breeding water and fed with a regular or HCD containing CholEsteryl BODIPY 542/563 C11 for 10 d. Finally, blood lipids of adult zebrafish fed with regular or HFD for seven weeks were measured. Oil Red O was not detected in the blood vessels of 7-8-dpf zebrafish. Increased intravascular lipid levels were detected in 7-dpf zebrafish incubated in 0.1% egg yolk, indicated by Oil Red O staining. Intravascular cholesterol was efficiently stained in 3-dpf zebrafish incubated in breeding water containing CholEsteryl BODIPY 542/563 C11; however, this method was inappropriate for the calculation of intravascular fluorescence intensity in zebrafish >7­dpf. In spite of this, intra-aortic fluorescence intensity of zebrafish fed a HCD containing CholEsteryl BODIPY 542/563 C11 was significantly higher (P<0.05) than that of those fed a regular diet containing CholEsteryl BODIPY 542/563 C11. The serum total cholesterol and triglyceride levels of adult zebrafish fed a HFD were markedly increased compared to those of the control group (P<0.05). In conclusion, the use of Oil Red O staining and CholEsteryl BODIPY 542/563 C11 may have applications in zebrafish intravascular lipid metabolism research and screens for novel lipid-regulating drugs.

Autophagy is involved in the cardioprotection effect of remote limb ischemic postconditioning on myocardial ischemia/reperfusion injury in normal mice, but not diabetic mice.

BACKGROUND: Recent animal study and clinical trial data suggested that remote limb ischemic postconditioning (RIPostC) can invoke potent cardioprotection. However, during ischemia reperfusion injury (IR), the effect and mechanism of RIPostC on myocardium in subjects with or without diabetes mellitus (DM) are poorly understood. Autophagy plays a crucial role in alleviating myocardial IR injury. The aim of this study was to determine the effect of RIPostC on mice myocardial IR injury model with or without DM, and investigate the role of autophagy in this process. METHODOLOGY AND RESULTS: Streptozocin (STZ) induced DM mice model and myocardial IR model were established. Using a noninvasive technique, RIPostC was induced in normal mice (ND) and DM mice by three cycles of ischemia (5 min) and reperfusion (5 min) in the left hindlimb. In ND group, RIPostC significantly reduced infarct size (32.6±3.0% in ND-RIPostC vs. 50.6±2.4% in ND-IR, p<0.05) and improved cardiac ejection fraction (49.70±3.46% in ND-RIPostC vs. 31.30±3.95% in ND-IR, p<0.05). However, in DM group, no RIPostC mediated cardioprotetion effect was observed. To analyze the role of autophagy, western blot and immunohistochemistry was performed. Our data showed that a decreased sequestosome 1 (SQSTM1/p62) level, an increased Beclin-1 level, and higher ratio of LC3-II/LC3-I were observed in ND RIPostC group, but not DM RIPostC group. CONCLUSIONS: The current study suggested that RIPostC exerts cardioprotection effect on IR in normal mice, but not DM mice, and this difference is via, at least in part, the up-regulation of autophagy.

The evaluation of rapid cooling as an anesthetic method for the zebrafish.

As zebrafish became a popular research system in contemporary biomedical research, effective anesthesia, which had low toxicity and high efficacy, was needed. The objective of this article was to evaluate the anesthetic effect of rapid cooling for embryo and larvae zebrafish with ice slush (ice and water admixture). The time to stage 5 anesthesia and maintaining for more than 5 s were detected and compared to MS-222 anesthesia. Besides, the time of recovery from anesthesia, mortality, and the survivability were measured and compared with MS-222 anesthesia. The results showed that anesthesia was generally achieved within 10 s for rapid cooling, which was more rapid than MS-222. The survivability assay demonstrated that rapid cooling was suitable for embryo and larvae zebrafish (1-14 days) and could be used for repeated anesthesia. The most important advantage was that this anesthesia could persist for 10 min and had no mortality. These findings suggested that rapid cooling provided advantages of improved safety, rapid anesthesia, and potentially low mortality rates and could be an effective anesthetic method for scientific research.

Effect of iron deficiency on c-kit⁺ cardiac stem cells in vitro.

AIM: Iron deficiency is a common comorbidity in chronic heart failure (CHF) which may exacerbate CHF. The c-kit⁺ cardiac stem cells (CSCs) play a vital role in cardiac function repair. However, much is unknown regarding the role of iron deficiency in regulating c-kit⁺ CSCs function. In this study, we investigated whether iron deficiency regulates c-kit⁺ CSCs proliferation, migration, apoptosis, and differentiation in vitro. METHOD: All c-kit⁺ CSCs were isolated from adult C57BL/6 mice. The c-kit⁺ CSCs were cultured with deferoxamine (DFO, an iron chelator), mimosine (MIM, another iron chelator), or a complex of DFO and iron (Fe(III)), respectively. Cell migration was assayed using a 48-well chamber system. Proliferation, cell cycle, and apoptosis of c-kit⁺ CSCs were analyzed with BrdU labeling, population doubling time assay, CCK-8 assay, and flow cytometry. Caspase-3 protein level and activity were examined with Western blotting and spectrophotometric detection. The changes in the expression of cardiac-specific proteins (GATA-4,TNI, and ß-MHC) and cell cycle-related proteins (cyclin D1, RB, and pRB) were detected with Western blotting. RESULT: DFO and MIM suppressed c-kit⁺ CSCs proliferation and differentiation. They also modulated cell cycle and cardiac-specific protein expression. Iron chelators down-regulated the expression and phosphorylation of cell cycle-related proteins. Iron reversed those suppressive effects of DFO. DFO and MIM didn't affect c-kit⁺ CSCs migration and apoptosis. CONCLUSION: Iron deficiency suppressed proliferation and differentiation of c-kit⁺ CSCs. This may partly explain how iron deficiency affects CHF prognosis.