Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

UNASSIGNED: Vascular calcification is a common clinical complication of chronic kidney disease (CKD), atherosclerosis (AS), and diabetes, which is associated with increased cardiovascular morbidity and mortality in patients. The transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteochondrogenic phenotype is a crucial step during vascular calcification. The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) plays an important role in regulating cell proliferation and differentiation, but whether it regulates the calcification of arteries and VSMCs remains unclear. Therefore, this study aims to understand the role of C/EBPα in the regulation of vascular calcification.
UNASSIGNED: Both mRNA and protein expression levels of C/EBPα were significantly increased in calcified arteries from mice treated with a high dose of vitamin D3 (vD3). Upregulation of C/EBPα was also observed in the high phosphate- and calcium-induced VSMC calcification process. The siRNA-mediated knockdown of C/EBPα significantly attenuated VSMC calcification in vitro. Moreover, C/EBPα depletion in VSMCs significantly reduced the mRNA expression of the osteochondrogenic genes, e.g., sex-determining region Y-box 9 (Sox9). C/EBPα overexpression can induce SOX9 overexpression. Similar changes in the protein expression of SOX9 were also observed in VSMCs after C/EBPα depletion or overexpression. In addition, silencing of Sox9 expression significantly inhibited the phosphate- and calcium-induced VSMC calcification in vitro.
UNASSIGNED: Findings in this study indicate that C/EBPα is a key regulator of the osteochondrogenic transdifferentiation of VSMCs and vascular calcification, which may represent a novel therapeutic target for vascular calcification.

Transcription factors and culture media were investigated to determine the condition to initiate the differentiation of human-induced pluripotent stem (iPS) cells most efficiently. The expression of genes in human adult liver was compared with that in 201B7 cells (iPS cells) using cDNA microarray analysis. Episomal plasmids expressing transcription factors were constructed. 201B7 cells were transfected with the episomal plasmids and cultured in ReproFF (feeder-free media maintaining pluripotency), Leibovitz-15 (L15), William\'s E (WE), or Dulbecco\'s modified Eagle medium/Nutrient F-12 Ham (DF12) for 7 days. RNA was isolated and subjected to real-time quantitative PCR to analyze the expression of alpha-feto protein (AFP) and albumin. cDNA microarray analysis revealed 16 transcription factors that were upregulated in human adult liver relative to that in 201B7 cells. Episomal plasmids expressing these 16 genes were transfected into 201B7 cells. CCAAT/enhancer-binding protein alpha (CEBPA), CCAAT/enhancer-binding protein beta (CEBPB), forkhead box A1 (FOXA1), and forkhead box A3 (FOXA3) up-regulated AFP and down-regulated Nanog. These four genes were further analyzed. The expression of AFP and albumin was the highest in 201B7 cells transfected with the combination of CEBPA, CEBPB, FOXA1, and FOXA3 and cultured in WE. The combination of CEBPA, CEBPB, FOXA1, and FOXA3 was suitable for 201B7 cells to initiate differentiation to the hepatocyte lineage and WE was the most suitable medium for culture after transfection. J. Cell. Biochem. 117: 2001-2009, 2016. © 2016 Wiley Periodicals, Inc.

OBJECTIVE: The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) has been shown to play an important role in liver development, cell proliferation and differentiation. It is, however, largely unknown if C/EBPα regulates cell differentiation and proliferation differently in the diverse cell types of the human liver. We investigated the role of C/EBPα in primary human fetal liver cells and liver cell subpopulations in vitro using a 3-D perfusion bioreactor as an advanced in vivo-like human organ culture model.
METHODS: Human fetal liver cells were investigated in vitro. C/EBPα gene expression was knocked down using siRNA or overexpressed by plasmid transfection. Cell type-specific gene expression was studied, cell populations and their proliferation were investigated, and metabolic parameters were analyzed.
RESULTS: When C/EBPα gene expression was knocked down, we observed a significantly reduced expression of typical endothelial, hematopoietic and mesenchymal genes such as CD31, vWF, CD90, CD45 and α-smooth muscle actin in fetal cells. The intracellular expression of hepatic proteins and genes for liver-specific serum proteins α-fetoprotein and albumin were reduced, their protein secretion was increased. Fetal endothelial cell numbers were reduced and hepatoblast numbers were increased. C/EBPα overexpression in fetal cells resulted in increased endothelial numbers, but did not affect mesenchymal cell types or hepatoblasts.
CONCLUSIONS: We demonstrated that the effects of C/EBPα are specific for the different human fetal liver cell types, using an advanced 3-D perfusion bioreactor as a human in vivo-like model.

MicroRNAs are small RNA molecules that post-transcriptionally regulate gene expression. MicroRNA-122 is the most abundant and specific liver microRNA. Hepatotoxicity involves a significant alteration of liver gene expression. The aim of this work was to evaluate the microRNA-122 regulatory network in models of hepatotoxicity induced by thioacetamide or carbon tetrachloride. We report that the toxins decreased the expression of microRNA-122, which corresponded with an increase in two target genes: Cyclin G1 and the cationic amino acid transporter CAT-1. We found a decreased expression of its precursor, pri-microRNA-122, and of the transcription factors that specifically bind its promoter: CCAAT/enhancer-binding protein alpha, and members of the hepatocyte nuclear factor family. Therefore, microRNA-122 expression levels are under transcriptional control during hepatotoxicity. We propose that the changes observed are associated with the liver response to cope with the injury caused by the hepatotoxins, likely through a cell proliferation process to repair the damaged tissue.

BACKGROUND: Hepatocellular carcinoma (HCC) associated to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths. Genetic variations may play an important role in the development of HCC in HCV patients. Ghrelin exerts anti-inflammatory, antifibrotic and hepatoprotective effects on chronically injured hepatic tissues. Ghrelin gene shows several single nucleotide polymorphisms (SNPs) including -604G/A, Arg51Gln, and Leu72Met. Hemochromatosis gene (HFE) mutations namely C282Y and H63D may cause hepatic iron overload, thus increasing the risk of HCC in HCV patients.
OBJECTIVE: To investigate the association of progression of HCC with ghrelin and HFE gene polymorphisms in HCV Egyptian patients.
METHODS: Seventy-nine chronic HCV patients (thirty-nine developed HCC and forty did not), and forty healthy control subjects were included in the study. The polymorphisms were evaluated by PCR/RFLP analysis, and related protein levels were measured by either ELISA or colorimetric assays.
RESULTS: The three tested SNPs on ghrelin gene were detected in the studied groups, only one SNP (Arg51Gln) showed significantly higher GA, AA genotypes and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC and controls. Of the two mutations studied on HFE gene only H63D heterozygous allele was detected, and its frequency did not statistically differ among studied groups.
CONCLUSIONS: Our results suggest that A allele at position 346 of the ghrelin gene is associated with susceptibility to HCC in hepatitis C patients.