m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.

Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2.

Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4(+)CD25(-) T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors.

Biological characteristics of the A1762T/G1764A mutant strain of hepatitis B virus in vivo.

The double nucleotide, A1762T and G1764A exchange (TA mutation), in the hepatitis B virus (HBV) genome basal core promoter (BCP) region is a common viral mutation in patients with chronic HBV infection. This mutation is located in the binding site of hepatocyte nuclear factor 4 (HNF4), and a number of liver­enriched transcription factors are involved in the regulation of HBV transcription and replication. The aim of the present study was to investigate the biological characteristics of the HBV strain with this mutation, and the effect of HNF4 inhibition on the replication of this strain in vivo. The results indicated that in vivo the HBV strain with the TA mutation supported a higher level of pregenomic RNA transcription and HBV DNA replication, compared with the wild­type strain. Furthermore, the concentration of serum HBeAg in the TA mutant group was lower than that in the wild­type strain. Following treatment of the mice with entecavir (ETV) or tenofovir disoproxil fumarate (TDF), the transcription and replication levels of wild­type and mutant strains were reduced. In the groups treated with TDF, the inhibition effect was more marked. In hepatocytes in which HNF4 expression was specifically inhibited, the level of 3.5 kb mRNA of HBV was reduced compared with that in mouse cells with normal HNF4 expression, and HBV DNA replication levels were also reduced to a greater extent. Furthermore, following liver­specific knockdown of HNF4, the reduction in variant virus expression was greater than that of the wild­type virus. In conclusion, the replication capacity of HBV with the TA mutation was increased, and the mutation was associated with a reduction in serum HBeAg levels. This mutant strain remained sensitive to ETV and TDF, and HNF4 supported a higher replication level of TA mutant HBV in vivo.

Role and functional domain of hepatitis B virus X protein in regulating HBV transcription and replication in vitro and in vivo.

The role of hepatitis B virus (HBV) X protein (HBx) in the regulation of HBV replication remains controversial. In the present study, the role of HBx in regulating HBV replication was initially investigated in both HepG2 and Huh7 in vitro cell lines with a transient transfection system. Next, the regions of HBx responsible for transcriptional transactivation and promotion of HBV replication were mapped in an HBV replication mouse model by in vivo transfection of a series of HBx expression plasmids. In an in vitro setting, HBx deficiency had little effect on HBV replication in Huh7 cells, but impaired HBV replication in HepG2 cells. In an in vivo setting, HBx had a strong enhancing effect on HBV transcription and replication. For the C-terminal two-thirds of the protein (amino acids [aa] 51 to 154) was required for this function of HBx, and the regions spanning aa 52 to 72 and 88 to 154 were found to be important for the stimulatory function of HBx on HBV replication. In conclusion, the role of HBx in HBV replication regulation is affected by host cell type, and HBx has an important role in stimulating HBV transcription and replication in hepatocytes in vivo. Further, the transcriptional transactivation function of HBx may be crucial for its stimulatory effect on HBV transcription and replication.

Inhibiting the expression of hepatocyte nuclear factor 4 alpha attenuates lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure in mice.

BACKGROUND: Hepatocyte nuclear factor 4 alpha (HNF4alpha) plays an important role in regulating cytokine-induced inflammatory responses. This study aimed to investigate the role of HNF4alpha in the development of fulminant hepatic failure (FHF) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN). METHODS: The FHF model was induced by simultaneous intraperitoneal injection of LPS/D-GalN in mice. Three days prior to LPS/D-GalN administration, HNF4alpha short-hairpin interfering RNA expression plasmid or physiological saline was injected via the tail vein with a hydrodynamics-based procedure. The degree of hepatic damage and cumulative survival rate were subsequently assessed. RESULTS: The expression of HNF4alpha was increased in the early stage after LPS/D-GalN administration. Inhibiting the expression of HNF4alpha reduced serum levels of alanine aminotransferase and aspartate aminotransferase, alleviated histological injury, and improved the survival of mice with FHF. In addition, both serum and hepatic tumor necrosis factor alpha expression were suppressed when HNF4alpha expression was inhibited in mice with FHF. CONCLUSION: Inhibiting HNF4alpha expression protects mice from FHF induced by LPS/D-GalN, but the exact mechanism behind this needs further investigation.