miR-365 regulates liver cancer stem cells via RAC1 pathway.

Liver cancer stem cells (CSCs) were involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). miR-365 was downregulated in hepatocellular carcinoma and inhibited HCC cell proliferation and invasion. However, the role of miR-365 in liver cancer stem cells was unknown. Herein, we observed a remarkable decrease of miR-365 expression in CD133 or EpCAM-positive liver CSCs as well as in CSC-enriched hepatoma spheres. Up-regulated miR-365 suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self-renewal ability of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in hepatoma cells downregulated the RAC1 mRNA and protein expression. RAC1 also could promote the expansion of liver CSCs. The special RAC1 inhibitor EHop-106 or RAC1 overexpression abolished the discrepancy in liver CSC proportion and the self-renewal capacity between miR-365 overexpression hepatoma cells and control cells, which further confirmed that RAC1 was required in miR-365-suppressed liver CSCs expansion. miR-365 was downregulated in liver CSCs and could inhibit HCC cells dedifferentiation and liver CSCs expansion by targeting RAC1 signaling.

Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis.

AIMS: To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC). MATERIALS AND METHODS: After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis. RESULTS: After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-ß (TGF-ß), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3. CONCLUSIONS: The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-ß and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.

Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway.

AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival. METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay. RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3' untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells. CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.

Inhibition of c-Myc by let-7b mimic reverses mutidrug resistance in gastric cancer cells.

Chemotherapy is one of the few effective choices for patients with advanced or recurrent gastric cancer (GC). However, the development of mutidrug resistance (MDR) to cancer chemotherapy is a major obstacle to the effective treatment of advanced GC. Additionally, the mechanism of MDR remains to be determined. In the present study, we tested IC50 of cisplatin (DDP), vincristine (VCR) and 5-fluorouracil (5-FU) in SGC7901, SGC7901/DDP and SGC7901/VCR gastric cancer cells using an MTT assay. The expression of let-7b and c-Myc in these cells was detected by qPCR and western blot analysis. The relationship between let-7b and c-Myc was explored using a luciferase reporter assay. Transfection of let-7b mimic or inhibitor was used to confirm the effect of let-7b on drug sensitivity in chemotherapy via the regulation of c-Myc expression. We found that the expression of let-7b was lower in chemotherapy-resistant SGC7901/DDP and SGC7901/VCR gastric cancer cells than that in chemotherapy-sensitive SGC7901 cells. By contrast, the expression of c-Myc was higher in SGC7901/DDP and SGC7901/VCR cells than that in SGC7901 cells. Furthermore, we confirmed that let-7b suppresses c-Myc gene expression at the mRNA and protein levels in a sequence-specific manner, while transfection of let-7b mimic increases drug sensitivity in chemotherapy-resistant SGC7901/DDP and SGC7901/VCR cells by targeting downregulation of c-Myc. In SGC7901 drug-sensitive cells, however, the sensitivity of chemotherapy was significantly decreased following let-7b inhibitor transfection. The present study results demonstrated that let-7b increases drug sensitivity in chemotherapy­resistant SGC7901/DDP and SGC7901/VCR gastric cancer cells by targeting the downregulation of c-Myc and that, let-7b mimic reverses MDR by promoting cancer stem cell differentiation controlled by double-negative autoregulatory loops (Lin28/let-7 and Myc/let-7) and a double-positive autoregulatory loop (Lin28/Lin28B/Myc) existing in GC cells, which remains to be confirmed.

Egg consumption and breast cancer risk: a meta-analysis.

The relationship between egg consumption and breast cancer risk has been inconsistent, so it is necessary to conduct a meta-analysis to evaluate the relationship. PubMed, EMBASE and ISI Web of Knowledge were searched to find cohort studies or case control studies that evaluated the relationship between egg consumption and breast cancer risk. A comprehensive meta-analysis software was used to conduct the meta-analysis. 13 studies were included. The meta-analysis results showed that egg consumption was associated with increased breast cancer risk (RR 1.04, 95 % CI 1.01-1.08). Subgroup analyses showed egg consumption was also associated with increased breast cancer risk based on cohort studies (RR 1.04, 95 % CI 1.00-1.08), among European population (RR 1.05, 95 % CI 1.01-1.09), Asian population (RR 1.09, 95 % CI 1.00-1.18), postmenopausal population (RR 1.06, 95 % CI 1.02-1.10), and those who consumed ≥2, ≤5/week (RR 1.10, 95 % CI 1.02-1.17), but not in case-control studies (RR 1.06, 95 % CI 0.97-1.15), among American population (RR 1.04, 95 % CI 0.94-1.16), premenopausal population (RR 1.04, 95 % CI 0.98-1.11) and those who consumed ≥1, <2/week (RR 1.04, 95 % CI 0.97-1.11) or >5 eggs/week (RR 0.97, 95 % CI 0.88-1.06). Egg consumption was associated with increased breast cancer risk among the European, Asian and postmenopausal population and those who consumed ≥2, ≤5/week.