Effect and Mechanism of LncRNA HOTAIR on Migration, Apoptosis and Proliferation of Hepatocellular Carcinoma Cells

<sec> <title>Objective:</title> This study was designed to probe the influence and mechanism of lncRNA HOTAIR on migration, apoptosis and proliferation of hepatocellular carcinoma (HCC) cells. </sec> <sec> <title>Methods:</title> We evaluated LncRNA HOTAIR expression in HCC tissues and adjacent tissues, and serum of HCC patients and healthy controls. Later, we knocked down lncRNA HOTAIR, and utilized CCK-8 to determine Hep3B cell proliferation, flow cytometry for prospecting Hep3B cell apoptosis, and cell scratch assay for observing Hep3B cell migration.We anticipated the direct target of lncRNA HOTAIR, and adopted luciferase reporter assay to verify. Moreover, we inhibitedmiR-126-5p expression, and rescue experiment for evaluating the influence of si-HOTAIR+miR-126-5p inhibitors on Hep3B cell migration, apoptosis as well as proliferation. </sec> <sec> <title>Results:</title> Our results showed that lncRNA HOTAIR expression in tumor tissues and serum was significantly increased. Moreover, lncRNA HOTAIR inhibition significantly decreased the Hep3B cell proliferation rate, elevated Hep3B cell apoptosis rate, and inhibited Hep3B cell migration. Luciferase reporter assay suggested that miR-126-5p was the direct target of lncRNA HOTAIR. Furthermore, co-transfection of si-HOTAIR+miR-126-5p inhibitor could diminishthe effects of HOTAIR silencing on apoptosis, proliferation and migration. </sec> <sec> <title>Conclusion:</title> Silencing of lncRNA-HOTAIR can inhibit the HCC cell migration and proliferation, and increase the apoptosis by up-regulating miR-126-5p expression. </sec>

Mutation of EPO 5`UTR Facilitates Interaction with HIF2 and Causes Autosomal Dominant Erythrocytosis

We studied 10 affected and 11 non affected relatives of a five generation kindred with autosomal dominant familial erythrocytosis. We have excluded other known inherited forms of erythrocytosis. i.e., mutations of globin, the 2,3 DPG generating PBGM gene causing increased Hb/O2 affinity (low p50), gain-of-function mutations of erythropoietin receptor (EPOR), germ-line JAK2 mutations, and hypoxia inducible factor 2A (HIF2-A(EPAS1)),PHD2(EGLN1), and VHL mutations associated with augmented oxygen-sensing pathway. Those affected family members had moderately increased erythropoietin (EPO) levels, no splenomegaly, normal leukocyte and platelet numbers and normal p50 (presented at this mtg, Blood. 2003;102,162b). We sequenced whole exomes and adjacent portions of introns of two affected individuals and found a novel heterozygous 5`UTR EPO variant with change -136 nt upstream from the ATG EPO initiation site (NG_021471 -136 G&gt;A). This variant segregated with the erythrocytosis phenotype in 15 relatives examined: the 7 affected subjects were heterozygous for this variant and the 8 unaffected were negative, suggesting its causative role in erythrocytosis (presented at this mtg, Blood. 2013;122,950). Other authors (NEJM 2018; 378:924) reported a variant of autosomal dominant familial erythrocytosis with a different EPO mutation: a single-nucleotide deletion (c.32delG) in exon 2 of the EPO gene causing a frameshift and alternative EPO mRNA transcripts, leading to increased production of functional EPO protein with shortened signal peptide and a novel N-terminus as cause of their familial erythrocytosis. In order to characterize function of our 5`UTR EPO variant, we introduced it into the EPO producing human hepatoma cell line Hep3B using CRISPR/Cas9 editing system by homologous recombination with single-stranded donor oligonucleotides. The targeted cells were sorted in 96 well plates (20 cells per well) and then each well tested for presence of -136 G&gt;A variant by allele-specific PCR. We identified 3 heterozygous Hep3B for EPO-136 G&gt;A; the second round of targeting generated homozygous Hep3B clones. The EPO mRNA of homozygous recombinants was greatly increased and detected even in normoxia, unlike non-edited Hep3B cells. No alternative EPO mRNA transcripts were detected in the engineered and non-edited Hep3B cells. To emulate human phenotype, the supernatants of cultured three heterozygousEPO-136 G&gt;A Hep3B single-cell derived clones and controls in normoxic and hypoxic conditions were used to detect production of EPO. The hypoxic treatment increased ~2x the production of EPO from edited clones compared to non-edited Hep3B cells. The secreted EPO from heterozygousEPO-136 G&gt;A Hep3B clones supported growth of EPO-dependent BaF3-EPOR cells more than supernatants from non-edited Hep3B cells. We then measured EPO transcript levels in Hep3B with EPO-136 G&gt;A and parental Hep3B cell lines in normoxia and hypoxia. The hypoxia increased the relative expression of EPO-136 G&gt;A allele in all three targeted heterozygousEPO-136 G&gt;A Hep3B cell lines. The EPO gene promoter was largely unmethylated in both wild and mutated clones. To evaluate activity of this mutant EPO promoter, we sub-cloned wild and mutated 5`UTR EPO sequence upstream of luciferase reporter gene and transfected them into two EPO producing cell lines - Hep3B, Hep2G. The mutant significantly increased activity of the reporter. To study the interaction of EPO-136 G&gt;A mutant promoter with HIF2 (principal transcription factor regulating EPO), we co-transfected EPO-luc reporter with HIF2-A expression plasmid. The activity of reporter with mutated EPO-136 G&gt;A was further increased in these cells with augmented HIF2 levels. Indeed, alignment tools predicted the EPO-136 G&gt;A genomic region as putative HIF2 binding site. This suggests that mutated 5`UTR of EPO augments interaction with HIF2, leading to increase production of EPO. Chromatin immunoprecipitation experiments are ongoing to model the transcriptional regulatory network accounting for augmented transcriptional regulation of this 5`UTR EPO gene variant. Here we report a novel mechanism of inherited erythrocytosis caused by increased transcription of mutated 5`UTR of EPO. Supported by Czech HRC, grant NV19-07-00412 and Ministry of Education, grant LTAUSA17142 and University of Utah. Disclosures No relevant conflicts of interest to declare.

Anticancer and Antiangiogenic Activities of Novel α-Mangostin Glycosides in Human Hepatocellular Carcinoma Cells via Downregulation of c-Met and HIF-1α

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In the present study, for the first time, we evaluated the anticancer and antiangiogenic activities of α-mangostin-3-O-β-D-2-deoxyglucopyranoside (Man-3DG) and α-mangostin 6-O-β-D-2-deoxyglucopyranoside (Man-6DG), glycosides of α-mangostin, against human HCC cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed the growth of three different HCC cells (Hep3B, Huh7, and HepG2) as well as the migration of Hep3B cells. Furthermore, they induced cell cycle arrest in the G0/G1 phases and apoptotic cell death by regulating apoptosis-related proteins of mitochondria in Hep3B cells. Noticeably, Man-3DG and Man-6DG also caused autophagy, while co-treatment of the α-mangostin glycosides with an autophagy inhibitor 3-MA enhanced the inhibitory effect on Hep3B cell growth in comparison to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway that plays a critical role in the pathogenesis of HCC. Furthermore, the α-mangostin glycosides decreased Hep3B cell-induced angiogenesis in vitro through the downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Notably, Man-6DG more effectively inhibited the growth, tumorsphere formation, and expression of cancer stemness regulators compared to α-mangostin and Man-3DG in 3D spheroid-cultured Hep3B cells. These findings suggest that the α-mangostin glycosides might be promising anticancer agents for HCC treatment with superior pharmacological properties than the parent molecule α-mangostin.

Chemical composition and cytotoxic activity of the essential oils of Cymbopogon citratus L. Grown in phu tho province

Culms and leaves of Cymbopogon citratus L. were collected from two regions of Phu Tho province (Thanh Son and Phu Ninh) and used as materials for essential oil extraction. Oils obtained were steam-distilled, analyzed for chemical composition and evaluated for cytotoxic activity against three different cancer cell lines. The GC/MS analysis showed that citral is the major content of the steam-distilled essential oils which was found in the range of 64.15-76.22%. Camphene was found only in culm oils of both regions but it was not detected in the leaf oils. Interestingly, the isomer forms of ocimene present at higher content in the culm oils than in the leaf oils whereas myrcene content in the leaf oils is higher than that in the culm oils. In a cytotoxicity test, four essential oils of culms and leaves of C. citratus from Thanh Son and Phu Ninh showed potent activity against A549 (human lung carcinoma) cell line with the IC50 values ranging from 4.01±0.39 to 6.3±0.54 µg/ml. The essential oils (culms and leaves) from Phu Ninh exhibited moderate effects on the Hela (human cervical adenocarcinoma) cells with the IC50 values of 19.43±1.16 and 42±2.41 µg/ml, respectively. However, they were inactive against the human hepatocellular carcinoma Hep3B cell line. The essential oils from Thanh Son exhibited potent cytotoxic activity against Hela and Hep3B cell lines with the IC50 values ranging from 1.18±0.26 to 8.91±0.32 µg/ml. The results indicated that the essential oils of C. citratus from Thanh Son, Phu Tho could be considered as a promising candidate for the natural sources of anticancer agents.

Disulfiram and disulfiram-loaded poly-[lactide-co-glycolic acid] nanoparticles modulate metastatic markers and proteasomal activity in hepatocarcinoma Hep3b cell line

Hepatocellular carcinoma (HCC) results in significantly high mortality rates due to its subtle metastatic expressions. Exorbitant costs of anticancer drugs have lead to the concept of repositioning standard drugs for their anticancer potential. One such antialcoholic drug, disulfiram (DSF), has been reported to show significant cytotoxicity (IC