Establishment of a Prognostic Model for Hepatocellular Carcinoma Based on Bioinformatics and the Role of NR6A1 in the Progression of HCC.

Background and Aims: Generally acceptable prognostic models for hepatocellular carcinoma (HCC) are not available. This study aimed to establish a prognostic model for HCC by identifying immune-related differentially expressed genes (IR-DEGs) and to investigate the potential role of NR6A1 in the progression of HCC. Methods: Bioinformatics analysis using The Cancer Genome Atlas and ImmPort databases was used to identify IR-DEGs. Lasso Cox regression and multivariate Cox regression analysis were used to establish a prognostic model of HCC. Kaplan-Meier analysis and the receiver operating characteristic (ROC) curves were used to evaluate the performance of the prognostic model, which was further verified in the International Cancer Genome Consortium (ICGC) database. Gene set enrichment analysis was used to explore the potential pathways of NR6A1. Cell counting kit 8, colony formation, wound healing, and Transwell migration assays using Huh7 cells, and tumor formation models in nude mice were conducted. Results: A prognostic model established based on ten identified IR-DEGs including HSPA4, FABP6, MAPT, NDRG1, APLN, IL17D, LHB, SPP1, GLP1R, and NR6A1, effectively predicted the prognosis of HCC patients, was confirmed by the ROC curves and verified in ICGC database. NR6A1 expression was significantly up-regulated in HCC patients, and NR6A1 was significantly associated with a low survival rate. Gene set enrichment analysis showed the enrichment of cell cycle, mTOR, WNT, and ERBB signaling pathways in patients with high NR6A1 expression. NR6A1 promoted cell proliferation, invasiveness, migration, and malignant tumor formation and growth in vitro and in vivo. Conclusions: An effective prognostic model for HCC, based on a novel signature of 10 immune-related genes, was established. NR6A1 was up-regulated in HCC and was associated with a poor prognosis of HCC. NR6A1 promoted cell proliferation, migration, and growth of HCC, most likely through the cell cycle, mTOR, WNT, and ERBB signaling pathways.

MiR-30b-5p regulates the lipid metabolism by targeting PPARGC1A in Huh-7 cell line.

BACKGROUND: MiRNAs are a group of multifunctional non-coding RNAs which play an important role in the various physiological processes including the development of NAFLD. Recent studies have shown that miR-30b-5p tightly associated with the abnormal lipid metabolism in patients with NAFLD, but the detailed mechanism of miR-30b-5p in the lipid metabolism was remain unclear. The aim of this study was to investigate the effect of miR-30b-5p on the lipid metabolism in hepatocellular carcinoma Huh-7 cells. MATERIAL AND METHODS: The correlation of intracellular fat content with the expression of miR-30b-5p in Huh-7 cells and HepG2 cells was investigated by treated cells with different concentrations of FFAs. The effect of miR-30b-5p on the lipid deposition in Huh-7 cells was tested by oil red O staining and TG concentrations measurement. qRT-PCR and western blot were used to investigate the lipid metabolism-related genes PPAR-α, SREBP-1, and GULT1 in miR-30b-5p overexpressed or inhibited Huh-7 cells. Target genes of miR-30b-5p were predicted using starBase, miRDB, and TargetScan databases and verified by qRT-PCR and western blot. RESULTS: The expression of miR-30b-5p was significant decreased in the FFAs treated Huh-7 cells and HepG2 cells. Overexpressing miR-30b-5p in Huh-7 cells decreased the number and size of lipid droplets and intracellular TG concentrations in Huh-7 cells. Expression of fatty acid oxidation related gene PPAR-α was increased and expression of lipid synthesis related gene SREBP-1 was decreased in the miR-30b-5p overexpressed Huh-7 cells. In addition, miR-30b-5p regulates the intracellular lipid metabolism by targeting PPARGC1A. CONCLUSIONS: Overexpression of miR-30b-5p could reduce the intracellular fat deposition in Huh-7 cells, and miR-30b-5p might regulate the intracellular lipid metabolism by targeting the PPARGC1A in Huh-7 cells.

A novel specific cleavage of IκBα protein in acute myeloid leukemia cells involves protease PR3.

IκBα protein plays an important role in NFκB signaling pathway regulation. The dysfunction of IκBα is tightly related to various diseases, including cancers. However, the molecular mechanisms by which IκBα loses its normal functions are diverse and complex. Here, we reported a novel cleavage of IκBα protein occurred in AML cells. Compared with the full-length IκBα protein, the truncated IκBα fragment exhibited a dramatically weak binding ability to NFκB complex and showed a significant decreased inhibition on NFκB transactivation. Knockdown of PR3, a serine protease mainly expressed in myeloid cells, could inhibit such IκBα cleavage and enhance the sensitivities of AML cells to the differentiation inducers. In addition, we showed that the level of PR3 mRNA was relatively higher in newly diagnosed AML patients than in those patients with complete remission, suggesting that PR3 expression and its involvement in IκBα cleavage might be closely associated with AML. Our studies revealed for the first time a PR3-involved IκBα cleavage in AML cells, providing some new evidences for further understanding the mechanisms underlying the deregulation of NFκB pathway in AML. Finally, we also suggested a potential clinical application value of PR3 protein in the treatment and prognosis surveillance for leukemia.

[Relationship of GSTT1 and GSTM1 Gene Polymorphisms and PAH-DNA Adducts with Pathogenesis of Multiple Myeloma].

OBJECTIVE: To explore the relationship of GSTT1, GSTM1 gene polymorphisms and PAH-DNA adduct with pathogenesis of multiple myeloma. METHODS: The bone marrow samples from 37 newly-diagnosed MM patients and 52 healthy peoples as controls were collected; the PCR-based restriction fragment length polymorphism (PCR-RFLP) method was used to detecte the polymorphism of GSTT1 and GSTM1, and to analysis their relationship with clinical characters of MM patients; the engyme linked immunosorbent assay (ELISA) was performed to detect the concentration of PAH-DNA adducts. RESULTS: GSTT1 null and GSTM1 null genotypes increased the risk of multiple myeloma with OR 2.57 (P=0.035) and 1.37 (P>0.05) respectively. In MM patients group, GSTT1 null genotype in stage III was significantly higher than that in stages I, II (P=0.038). However, no statistically significant association was found between GSTT1 gene polymorphism and clinical characters, such as age, type, hemoglobin, ß2-MG, albumin. Compared with Hb≥85 g/L of the newly-diagnosed MM patients, MM patients with Hb<85 g/L had significantly higher incidence of GSTM1 null genetype (P<0.05). The level of PAH-DNA adducts in MM patients was higher than that in controls (2358±1182 pg/ml vs 1853±996 pg/ml) (P<0.05). GSTT1 null genotype with PAH-DNA level≥2100 pg/ml showed a risk index of MM (OR=4.500, P<0.05). CONCLUSION: GSTT1 gene may have a critical function in the development of MM, and correlates with staging of MM. GSTM1 gene polymorphism correlates with hemoglobin levels of patients with MM. The content of PAH-DNA adducts may play an important role in the pathogenesis of multiple myeloma.

miR-141 confers docetaxel chemoresistance of breast cancer cells via regulation of EIF4E expression.

Resistance to docetaxel, a chemotherapy drug for breast cancer (BC) treatment, occurs in ~50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through miR-141 has been proven to play an important role in cancer drug resistance. The present study investigated the role of miR-141 expression in BC cells of acquired docetaxel resistance. Inhibition of miR-141 enhanced the response to docetaxel in docetaxel-resistant cells (MCF-7/DTX and MDA-MB-231/DTX, respectively), whereas overexpression of miR-141 confered resistance in docetaxel-sensitive cells (MCF-7 and MDA-MB-231, respectively). By directly targeting the eukaryotic translation initiation factor 4E (EIF4E) mRNA, miR-141 acts on genes that are necessary for drug induced apoptosis rendering the cells drug resistant. Modulation of miR-141 expression was correlated with EIF4E expression changes and a direct interaction of miR-141 with EIF4E was shown by a luciferase assay. Thus, the present study is the first to show an increased expression of miR-141 in an acquired model of docetaxel resistance in BC. This serves as a mechanism of acquired docetaxel resistance in BC cells, possibly through direct interactions with EIF4E, therefore presenting a potential therapeutic target for the treatment of docetaxel resistant BC.

MOR1 expression in gastric cancer: a biomarker associated with poor outcome.

BACKGROUND: At present, the expression of MOR1 and its function in gastric cancer remains unclear with evidence suggesting that it is to be involved in tumor progression and metastasis. The study was to assess the clinicopathologic relevance and prognostic value of MOR1 expression in gastric cancer. METHODS: Real-time quantitative RT-PCR and immunohistochemical staining were used to detect MOR1 expression in primary gastric cancerous surgical specimens and adjacent nontumorous tissues. RESULTS: High MOR1 expression was detected in cancerous tumor compared with their adjacent nontumorous tissues. In addition, the chi-square test revealed that high MOR1 expression was significantly correlated with depth of invasion (p = 0.006), lymph node metastasis (p = 0.001), distant metastasis (p = 0.017), and TNM staging (p = 0.027). Moreover, Kaplan-Meier analysis revealed a significant association between MOR1 expression and overall survival. High expression of MOR1 was identified as an independent and significant predictor gene of reduced postoperative survival. CONCLUSION: We conclude that MOR1 expression may be a useful biomarker for better prediction of the clinical outcome and management of gastric cancer patients.

Prognosis significance of HER2 status and TACC1 expression in patients with gastric carcinoma.

HER2 amplification and/or expression occurs in gastric carcinoma (GC), but the role of HER2 in the prognosis of GC remains unclear. The dysregulation of transforming acidic coiled coil 1 (TACC1), a downstream gene of HER2, is thought to be involved in the development of GC. The aim of this study was to investigate the role and relationship of HER2 and TACC1 in GC. The expression of HER2 and TACC1 was analyzed using immunohistochemistry on 129 primary resected GC patients, and HER2 amplification was additionally determined by FISH. The data on clinicopathological features and relevant prognostic factors in these patients were analyzed. The expression (3+, 2+ and 1+) and the amplification of HER2 was observed in 57 cases (44.2 %) and 25 cases (19.4 %), respectively, and the correlation between HER2 expression and amplification was strong (p < 0.001). According to the FDA criteria, 24 cases (18.6 %) would have been considered as HER2 positive. A total 62 (48.1 %) GC tissues showed positive cytoplasmic staining of TACC1. There was a significant and positive association between TACC1 and HER2. HER2 positive was significantly associated with TNM stage (p = 0.019), and TACC1 expression was significantly associated with lymph node metastasis (p = 0.004) and TNM stage (p = 0.004). TNM stage, TACC1 expression and co-positive of both HER2 and TACC1 were independent prognostic factors. TACC1 expression is an independent prognostic indicator of GC. The correlation between TACC1 expression and HER2-positive status indicated a possible synergistic regulation of the two molecules and co-positive of both HER2 and TACC1 maybe a more valuable prognostic marker.

Rig-G negatively regulates SCF-E3 ligase activities by disrupting the assembly of COP9 signalosome complex.

We previously showed that Rig-G, an antiproliferative protein induced by interferon, can sequester CSN5 protein in the cytoplasm. Here, we report that Rig-G can destroy the functions of CSN5-containing COP9 signalosome (CSN), a highly conserved multiprotein complex implicated in protein deneddylation, deubiquitination, and phosphorylation. By damaging integrity and stability of the CSN complex, Rig-G can dramatically reduce the cellular content of CSN complex and inhibit its regulatory roles in assembly and activation of cullin-RING ubiquitin E3 ligases (CRL). Furthermore, Rig-G can cause excessive activation of CRL through inhibition of CSN-mediated deneddylation, largely decreasing protein levels of Cul1 and ßTrCP, two important subunits of SCF (Skp1-Cul1-F-box protein)-E3 ligase. Rig-G can also attenuate the ability of CSN to recruit USP15 and impair CSN-associated deubiquitination. Increased autoubiquitination of ßTrCP and concomitant accumulation of target substrates (such as IκBα) are observed in Rig-G-expressing cells. Taken together, our findings reveal for the first time the negative regulation of Rig-G on SCF-E3 ligase activities through disrupting CSN complex, not only contributing to further investigation on biological functions of Rig-G, but also leading to better understanding of the CSN complex as a potential target in tumor diagnosis and treatment.

[Role of auto-secreted interferon α in all-trans retinoic acid-induced expression of RIG-G gene].

OBJECTIVE: To explore the relationship between interferon (IFN) α and all-trans retinoic acid (ATRA)-induced signaling pathways in the expression of retinoic acid-induced gene G (RIG-G). METHODS: Acute promyelocytic leukemia cell line NB4 and signal transducer and activator of transcription (STAT)1-deficient U3A cells were used. The protein levels of tyrosine-phosphorylated STAT2 in ATRA-treated NB4 cells were detected by Western blot. The culture supernatants of NB4 cells treated with ATRA for different time or U3A cells transfected with interferon regulatory factor (IRF)-1 were respectively collected. And the concentration of IFN-α was determined by enzyme-linked immunosorbent assay (ELISA). The effects of NB4 cell culture supernatants on the phosphorylation of STAT2 and the expression of RIG-G were detected by Western blot. RESULTS: The level of phosphorylated-STAT2 was obviously up-regulated in NB4 cells treated with ATRA for 72 hours, as well as the concentration of IFN-α in culture supernatants. The concentration of IFN-α increased from (1.5 ± 0.5) pg/ml in the untreated group to (7.6 ± 0.3) pg/ml (P < 0.05). After a 96-hour treatment, the concentration of IFN-α was up to (63.8 ± 5.8) pg/ml. And these culture supernatants could induce the tyrosine phosphorylation of STAT2 and up-regulate the protein level of RIG-G. As for U3A cells transfected with IRF-1, the concentration of IFN-α from the culture supernatant also increased 3-fold versus the control group transfected with empty vectors [(8.8 ± 1.4) pg/ml vs (3.4 ± 0.4) pg/ml, P < 0.05]. CONCLUSIONS: RIG-G gene expression is closely correlated with the cross-talk between ATRA and IFN-α-induced signaling pathways. ATRA increases the secretion of IFN-α by up-regulating the protein level of IRF-1. Then the secreted IFN-α may induce the phosphorylation of STAT2 and reinforce the expression of RIG-G.