Role of the 3' untranslated region in the post-transcriptionnal regulation of KLF6 gene expression in hepatocellular carcinoma

Krüppel-like factor 6 (KLF6) a été identifié comme gène suppresseur de tumeur impliqué dans le contrôle de la croissance, de la différentiation cellulaire et dans la mort par apoptose. L'expression de KLF6 est altérée au cours de la carcinogenèse hépatique, et cette protéine joue un rôle prépondérant dans le contrôle de la prolifération des cellules tumorales hépatiques. Nous avons étudié le rôle de la région 3'UTR de l'ARNm de KLF6 dans la régulation de son expression. Nous avons établi que la demi-vie de l'ARNm de KLF6 est fortement diminuée dans les lignées cellules dérivées du foie. La région 3'UTR inhibe fortement l'expression et l'activité de gènes rapporteurs en corrélation avec la région entre les nucléotides 1835 et 2615.Krüppel-like factor 6 (KLF6) is a tumor suppressor gene that is involved in the regulation of cell cycle, cellular proliferation, differentiation and cell death by apoptosis. KLF6 expression is altered in liver carcinogenesis. However, little is known on the mechanisms governing KLF6 expression in HCC. In the current study, we asked whether 3’UTR may be responsible for down regulation of KLF6 mRNA in HCC. Our results demonstrated that KLF6 mRNA half-life was greatly decreased in cells derived from HCC. Moreover, 3'UTR strongly inhibited the activity and expression of the reporter gene. In addition, we found that most of KLF6 3’UTR destabilisation activity resides between nucleotides 1835 and 2615

Dexamethasone improves the responsiveness of hepatoma cells for both free and solvent containing paclitaxel in vitro

This work was designated to explore the effect of glucocorticoid (dexamethasone) on the responsiveness of hepatoma cells to Paclitaxel (PTX) and the expression of Taxol resistance gene (Txr1) and the paclitaxel metabolizing genes. Hepatocellular carcinoma cells (HepG2) were treated with standard paclitaxel (PTX) or solvent containing paclitaxel (Taxol) in the presence or absence of dexamethasone (DEX). Cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Also, total RNA was isolated, reverse transcribed and used to determine the expression levels of Txr1, CYP 3A4, and CYP2C8 genes.Initially, HepG2 cells were more resistant to PTX than Taxol. Also, cells became more responsive to the standard PTX and Taxol in the presence of DEX, where the IC50 values decreased from 42.5 μg/ml to 13.07 μg/ml and from 6.5 μg/ml to 3.6 μg/ml, respectively. Apoptosis was the main mechanism of cytotoxicity in cells treated with PTX or Taxol. The involvement of DEX, however, decreased the percent of apoptotic cells. Moreover, the expression of Txr1 decreased by 18% and 35% in cells cotreated with PTX+DEX or Taxol+DEX. In parallel, the expression of paclitaxel metabolizing genes (CYP3A4 and CYP2C8) was increased compared to DEX free cells. This in vitro study reports the associations between the enhanced responsiveness of hepatoma cells to paclitaxel or Taxol in presence of dexamethasone, associated with a decrease in drug resistance and upregulation of the paclitaxel metabolizing genes.Keywords: Liver cancer, Paclitaxel nanoparticles, Taxol resistance gene, CYP

Unleash Multifunctional Role of miRNA Biogenesis Gene Variants (<i>XPO5*rs34324334</i> and <i>RAN*rs14035</i>) with Susceptibility to Hepatocellular Carcinoma

Numerous reports have explored the roles of different genetic variants in miRNA biogenesis mechanisms and the progression of various types of carcinomas. The goal of this study is to explore the association between XPO5*rs34324334 and RAN*rs14035 gene variants and susceptibility to hepatocellular carcinoma (HCC). In a cohort of 234 participants (107 HCC patients and 127 unrelated cancer-free controls) from the same geographic region, we characterized allelic discrimination using PCR-RFLP and performed subgroup analysis and multivariate regression. We found that the frequency of the XPO5*rs34324334 (A) variant was correlated with elevated risk of HCC under allelic (OR = 10.09, p-value p-value p-value p-value = 0.012), ascites (p-value = 0.003), and higher levels of alpha-fetoproteins (p-value = 0.011). Carriers of the RAN*rs14035 (T) variant were more likely to develop HCC under allelic (OR = 1.76, p-value = 0.003) and recessive (OR = 3.27, p-value XPO5*rs34324334 and RAN*rs14035 variants are independent risk factors for developing HCC

The Role of the 3' Untranslated Region in the Post-Transcriptional Regulation of KLF6 Gene Expression in Hepatocellular Carcinoma

KLF6 is ubiquitously expressed in human tissues and regulates many pathways such as differentiation, development, cellular proliferation, growth-related signal transduction, and apoptosis. We previously demonstrated that KLF6 expression is altered during liver carcinogenesis. More importantly, KLF6 invalidation results in cell cycle progression inhibition and apoptosis of liver cancer cells. On the other hand, enforced expression of KLF6 variant 2 (SV2) induces cancer cell death by apoptosis. Thus, we and others demonstrated that KLF6 and its splicing variants play a critical role in liver cancer. However, little is known on the mechanisms governing KLF6 expression in HCC. In the present work, we asked whether the 3' untranslated region (3'UTR) of the KLF6 mRNA may be responsible for regulation of KLF6 expression in HCC. We found that KLF6 mRNA stability was altered in liver-derived cell lines as compared to cervical cancer-derived cell lines and human embryonic fibroblasts. Interestingly, KLF6 mRNA was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes. We next cloned the KLF6 mRNA 3'UTR into luciferase-expressing vectors and found that gene expression and activity were strongly impaired in all liver-derived cell lines tested. In addition, we found that most the KLF6 3'UTR destabilisation activity resides between nt 1,835 and nt 2,615 of the KLF6 gene. Taken together, we provide the first steps towards better understanding of the regulation of KLF6 expression in HCC. Further work is needed to identify the factors that bind to KLF6 3'UTR to regulate its expression in liver cancer-derived cell lines

Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis&ndash;Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc