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Development of chemoresistance remains a major challenge in treating esophageal squamous cell carcinoma (ESCC) patients despite treatment advances. However, the role of RAC1 in chemoresistance of ESCC and the underlying mechanisms remain largely unknown. In this study, we found that higher levels of RAC1 expression were associated with poorer prognosis in ESCC patients. Enhanced RAC1 expression increased cell proliferation, migration, and chemoresistance in vitro. Combination therapy using RAC1 inhibitor EHop‐016 and cisplatin significantly promoted cell viability inhibition, G2/M phase cycle arrest, and apoptosis when compared to each monotherapy. Mechanistically, glycolysis was significantly downregulated in the RAC1 inhibitor monotherapy group and the combination group via inhibiting AKT/FOXO3a signaling when compared to the control group. Moreover, the silencing of RAC1 inhibited AKT/FOXO3a signaling and cell glycolysis while the upregulation of RAC1 produced an opposite effect. In murine xenograft models, the tumor volume and the expression of glycolytic enzymes were significantly reduced in combination therapy when compared to each monotherapy group. Overall, our study demonstrates that targeting RAC1 with an inhibitor overcomes cisplatin resistance in ESCC by suppressing glycolytic enzymes, which provides a promising strategy for treatment of ESCC in clinical practice. Glycolysis is essential for chemoresistance, anti‐apoptotic signaling, proliferation and migration of cancer cells. Our study reveals the role of RAC1 in regulating glycolytic enzymes. At the molecular level, RAC1 inhibitor suppresses GEF–RAC1 interaction to decrease AKT phosphorylation, which inhibits phosphorylation of FOXO3a and S6, and subsequently downregulates glycolytic enzymes such as PKM, LDHA, and HK1.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease, characterized by the excess accumulation of lipids in the liver. It has been demonstrated that the dysregulation of lipid droplet (LD)-associated proteins may be involved in the development of NAFLD. Adipose differentiation-related protein (ADRP), as one of the major LD-associated proteins, is expressed in normal and steatotic livers; however, the exact role of ADRP in the liver remains unknown. Previous studies have indicated that metformin, as an antidiabetic drug, effectively ameliorates NAFLD. However, its cellular and molecular mechanisms of action remain to be elucidated. Therefore, the aim of this study was to determine the role of ADRP in the metformin-mediated regulation of hepatic steatosis. We examined the effects of meformin in vivo and in vitro using ob/ob mice and primary hepatocytes, respectively. Lipid accumulation in the hepatocytes was induced by treatment with oleate. Our results revealed that metformin prevented hepatic steatosis in ob/ob mice and inhibited oleate-induced lipid accumulation in primary hepatocytes. Furthermore, using real-time PCR and western blot analysis, we examined the mRNA and protein expression of ADRP, respectively. We found that metformin significantly decreased the expression levels of ADRP. In addition, to further clarify the role of ADRP in lipid accumulation, we generated recombinant adenoviruses to induce the overexpression of ADRP and to knockdown ADRP. In the hepatocytes in which ADRP was overexpressed, the reducing effects of metformin on lipid accumulation were diminished. However, the knockdown of ADRP using siRNA targeting ADRP reduced the accumulation of triglycerides. Taken together, our data demonstrate that metformin prevents hepatic steatosis by regulating the expression of ADRP, which may be a key target in the treatment of NAFLD.

Cancer stem cells (CSCs) play important roles in the biological behaviour of malignant tumours. To study their properties, they must be carefully identified and purified. Cancer cells can acquire three different morphological types during single cell cloning. A small subpopulation of clones acquires a regular and compact shape, and these clones are enriched for CSCs; however, the majority of clones have an irregular morphology with loose intercellular junctions, with fewer characteristics of CSCs. At present, the main method to isolate CSCs is to collect the regular clones in low-density culture conditions; therefore, an insufficient amount of CSCs is obtained for clonal expansion. To obtain a more sufficient amount of CSCs, the clones with an irregular and loose morphology were examined in our study. We found a small subpopulation of U251 glioma cells that arrested in the suspended state and that subsequently migrated to form new clones. The suspended cells were isolated from the irregular and loose clones. Clonogenic assays were performed in which 43.70% of the suspended cells and 32.91% of the adherent cells formed new clones. To determine the biological differences between the suspended and adherent cells, carboxyfluorescein succinimidyl ester (CFSE) labelling, MTT assays, and cell cycle assays were performed. The results demonstrated that the suspended cells had the characteristics of CSCs, including higher proliferation rates, as well as self-maintenance and self-renewal capabilities, and they stained positively for markers of brain CSCs and had multilineage potential. Thus, we established a new and efficient approach for screening CSCs from the U251 human glioma cell line based on the cell growth state.

microRNA (miRNA) dysfunction is believed to play important roles in human diseases, including viral infectious diseases. Hepatitis C virus (HCV) infection promotes the development of steatosis, cirrhosis and hepatocellular carcinoma, which is genotype-specific. In order to characterize the miRNA expression profile of Huh-7 cells expressing the HCV core 3a vs. 1b, microarrays and real-time PCR were performed. Consequently, 16 miRNAs (5 miRNAs upregulated and 11 miRNAs downregulated) were found to be dysregulated. In addition, we generated the predicted and validated targets of the differentially expressed miRNAs and explored potential downstream function categories and pathways of target genes using databases of Gene Ontology (GO) and PANTHER and the database for annotation, visualization and integrated discovery (DAVID). The computational results indicated that the dysregulated miRNAs might perform the functions of cellular metabolism and cellular growth. Finally, these biological effects were preliminarily validated. This study identifies a specific miRNA expression profile in cells expressing HCV core proteins of different genotypes (genotype 3a and 1b), which may account for the variable pathophysiological manifestation associated with HCV infection.

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatic steatosis in patients with chronic HCV infection. Treatment of C57BL/6 mice infected with HCV core recombinant adenoviruses with resveratrol significantly decreased hepatic triacylglycerols (TAG) while the serum TAG level was unaffected. RT-PCR and Western blotting showed that HCV core protein attenuated the expression of Sirt1 and PPAR-α, which would be reversed by resveratrol. This was also confirmed in primary mouse hepatic cells infected with HCV core protein expressing adenovirus. Thus, resveratrol may prevent against hepatic steatosis by blocking the inhibited expression of Sirt1 and PPAR-α induced by HCV core protein.

ABSTRACT To understand the DNA-methylation mediated gene silencing mechanisms, we analyzed in cell culture of the promoter function of the MAGE-A1 gene, which is frequently demethylated and over-expressed in human hepatocellular carcinoma. We have established the correlation of the DNA methylation of the promoter CpG island with expression status of this gene in a panel of the established liver cancer cell lines. The crucial CpG dinucleotide(s) within the minimal promoter subjected to the control mediated by DNA methylation with profound biological functions was also delineated. Furthermore, a novel sequence-specific DNA-protein interaction at the −30 CpG dinucleotide upstream of the gene was found having a vital part to play in the DNA methylation mediated transcription silencing of the MAGE-A1 gene. Our results would not only provide new insights into the DNA methylation mediated mechanisms over transcription of the MAGE-A1 gene, but also pave the way for further defining the cross-talk among DNA methylation, histone modification and chromatin remodeling in detail.

The increasingly serious problem of acid rain is leading to increased potassium （K） loss from soils, and in our field investigation, we found that even congenerically relative Mosla species show different tolerance to K-deficiency. A hydroponic study was conducted on the growth of two Mosla species and their morphological, physiological and stoichiometric traits in response to limited （0.35 mmol K/L）, normal （3.25 mmol K/L） and excessive （6.50 mmol K/L） K concentrations. Mosla hangchowensis is an endangered plant, whereas Mosla dianthera a widespread weed. In the case of M. hangchowensis, in comparison with normal K concentration, K-limitation induced a significant reduction in net photosynthetic rate （Pn）, soluble protein content, and superoxide dismutase （SOD） actix, ity, but an increase in malondialdehyde （MDA） concentration. However, leaf mass ratio （LMR） and root mass ratio （RMR） were changed little by K-limitation. In contrast, for M. dianthera, K-limitation had little effect on Pn, soluble protein content, SOD activity, and MDA concentration, but increased LMR and RMR. Critical values of N （nitrogen）：K and K：P （phosphorus） ratios in the shoots indicated that limitation in acquiring K occurred under K-limited conditions for M. hangchowensis but not for M. dianthera. We found that low K content in natural habitats was a restrictive factor in the growth and distribution of M.. hangchowensis, and soil K-deficiency caused by acid rain worsened the situation of M. hangchowensis, while M. dianthera could well acclimate to the increasing K-deficiency. We suggest that controlling the acid rain and applying K fertilizers may be an effective way to rescue the endangered M. hangchowensis.

The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a region displaying a high score of loss of heterozygosity within chromosome 17p13.3 in human hepatocellular car-cinoma in China[l]. To unveil the underlying mechanisms for the transcription regulation of this gene and understand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by both bioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 gene was evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its tran-scription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragment containing the transcription start, extending from -112 to -55, represents the minimal promoter; 4, The consensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of the minimal promoter; 5, The bioinformatic analysis suggests that the C17ort25 gene may encode a protein in the family of the glyoxalase. Our data has provided some deep insight into both function and regulation of the C17orf25 gene in the context of the normal liver and hepatocellular carcinoma.

The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17p13.3 that suffers from a high frequent loss of heterozygosity in human hepatocellular carcinoma in China[1-5]. To understand the molecular mechanisms underlying transcription control of the RNMTL1 gene in human cancers, we decline using of the conventional approach where the cis-elements bound by the known transcription factors are primary targets, and carried out the systematic analyses to dissect the promoter structure and identify/characterize the key cis-elements that are responsible for its strong expression in cell. The molecular approaches applied included 1, the primer extension for mapping of the transcription starts; 2, the transient transfection/reporter assays on a large number of deletion and site-specific mutants of the promoter segment for defining the minimal promoter and the crucial elements within; and 3, the electrophoresis mobility shift assay with specific antibodies for reconfirming the nature of the transcription factors and their cognate cis-elements. We have shown that the interaction of an ATF/CREB element (-38 to -31) and its cognate transcription factors play a predominant role in the promoter activity of the RNMTL1 gene. The secondary DNA structures of the ATF/CREB element play a more vital role in the protein-DNA interaction. Finally, we reported a novel mechanism underlying the YY1 mediated transcription repression, namely, the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.