The effect of Epitranscriptomic writer (METTL3) silencing on Hepatocellular carcinoma cell line

Worldwide, HCC is the sixth most common malignancy and the third most common cause of cancer-related death. RNA epigenetics becomes a hot topic in recent years. Among more than a hundred different RNA modifications, m6A is the most abundant modification. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. METTL3 is a major RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through m6A modification. We aimed to target METTL3 in HepG2 cell lines by siRNA (small interfering RNA), and then evaluated the effect of this interference on viability and proliferative activity of HepG2 cells. Material and methods Using HepG2 cell lines, METTL3 was targeted using siRNA. The viability of HepG2 was conducted by Trypan blue exclusion test. The cell proliferation was tested by CellTiter 96® AQueous One Solution Cell Proliferation Assay. Results viable cell number and viability percent were significantly reduced in HepG2 cells transfected with siMETTL3 compared to mock cell lines (treated with transfection reagent only) (p<0.05). The active proliferative cell count was lower in cells transfected with siMETTL3 than mock cells (p<0.05). Conclusions Knockdown of METTL3 in HepG2 cell lines successively reduced cell viability and active proliferative cell count. METTL3 may be involved in liver tumorigenesis and its targeting may be of therapeutic benefit.

Phloretin Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rats by Regulating the Inflammatory Response, Oxidative Stress and Apoptosis

The inflammatory process is proposed to be one of the factors to benign prostatic enlargement (BPH), and this is the first study examining the anti-inflammatory ability of phloretin in treating rats with testosterone-induced BPH. BPH would be induced by testosterone (10 mg/kg/day testosterone subcutaneously for 28 days), and the other groups of rats were treated with phloretin 50 mg/kg/day or 100 mg/kg/day orally (phr50 or phr100 group) after induction. Prostate weight and prostate weight to body weight ratio were significantly reduced in the Phr100 group. Reduced dihydrotestosterone without interfering with 5α-reductase was observed in the phr100 group. In inflammatory proteins, reduced IL-6, IL-8, IL-17, NF-κB, and COX-2 were seen in the phr100 group. In reactive oxygen species, malondialdehyde was reduced, and superoxide dismutase and glutathione peroxidase were elevated in the phr100 group. In apoptotic assessment, elevated cleaved caspase-3 was observed in rats of the phr100 group. Enhanced pro-apoptotic Bax and reduced anti-apoptotic Bc1-2 could be seen in the phr100 group. In histological stains, markedly decreased glandular hyperplasia and proliferative cell nuclear antigen were observed with reduced expression in the phr100 group. Meanwhile, positive cells of terminal deoxynucleotidyl transferase dUTP nick end labeling were increased in the phr100 group. In conclusion, the treatment of phloretin 100 mg/kg/day could ameliorate testosterone-induced BPH.

Proliferative Activity of Myoepithelial Cells in of Mucoepidermoid Carcinoma

AIM: The aim of the study is to investigate the role of myoepithelial cells in the pathogenesis of mucoepidermoid carcinoma (MEC) using the double immunohistochemical staining; α _smooth muscle actin (_α-SMA)as specific marker for the myoepithelial cell differentiation and proliferative cell nuclear antigen (PCNA) as a marker for proliferative activity of myoepithelial cells. MATERIAL AND METHODS: Retrospective study of twenty salivary gland specimens (ten MEC and ten normal salivary glands) were studied using double immunohistochemical labeling for α _smooth muscle actin α-SMA) and proliferative cell nuclear antigen (PCNA). The SPSS statistical package was used for data analysis (IBM SPSS Statistics for Windows, Version 20.0, Released 2011, IBM Corp, and Armonk, NY, USA). RESULTS: In mucoepidermoid carcinomas, no positivity of α-SMA was seen in neoplastic cells (Frequent test), and it was just observed in the stroma of tumor, in the walls of blood vessels whereas, PCNA was positive, especially in high-grade tumors. In contrast, in normal salivary glands, the proliferating myoepithelial cells are stained by both α-SMA and PCNA. CONCLUSIONS: We believe that the myoepithelial cells have no a role in the development of mucoepidermoid carcinoma.

Gasotransmitter CO Attenuates Bleomycin-Induced Fibroblast Senescence via Induction of Stress Granule Formation

Cellular senescence is recognized as a phenomenon wherein a proliferative cell undergoes a permanent growth arrest. The accumulation of senescent cells over time can become harmful and result in diseases and physiological decline. Plasminogen activator inhibitor (PAI-1) is considered as a critical marker and mediator of cellular senescence. The formation of stress granules (SGs) could prevent senescence through the sequestration of PAI-1, and we previously suggested that exogenous carbon monoxide (CO) could induce SG assembly via integrated stress response (ISR). Although CO is known to possess anti-inflammatory, antioxidative, and antiapoptotic properties, whether it exerts antisenescent effect is still not well defined. Here, to address whether CO-induced SGs could protect against cellular senescence, we first treated lung fibroblasts with bleomycin (BLM) to establish DNA damage-induced cellular senescence, and observed a significant increase of several hallmarks of senescence through SA-β-gal staining, immunofluorescence, qRT-PCR, and Western blot assay. However, pre- and posttreatment of CO could remarkably attenuate these senescent phenotypes. According to our immunofluorescence results, CO-induced SGs could inhibit BLM-induced cellular senescence via sequestration of PAI-1, while it was abolished after the cotreatment of ISR inhibitor (ISRIB) due to the inhibition of SG assembly. Overall, our results proposed a novel role of CO in suppressing bleomycin-induced lung fibroblast senescence through the assembly of SGs.

MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis

There is so far no vaccine approved for human leishmaniasis, mainly because of the lack of appropriate adjuvants. This study aimed to evaluate in mice the capacity of a mixture of monophosphoryl lipid A (MPLA) and AddaVax® adjuvants in enhancing the efficacy of a Leishvacin®-like vaccine comprised of Leishmania amazonensis whole antigens (LaAg). For that, mice were immunized with LaAg plus MPLA/AddaVax® by the intramuscular route (i.m.) prior to challenge with 2 × 105 and 2 × 106 living parasites. Immunization with LaAg alone reduced the lesion growth of the 2 × 105-challenged mice only in the peak of infection, but that was not accompanied by reduced parasite load, and thus not considered protective. Mice given a 2 × 106 -challenge were not protected by LaAg. The association of LaAg with MPLA/AddaVax® was able to enhance the cutaneous hypersensitivity response compared with LaAg alone. Despite this, there was no difference in proliferative cell response to antigen ex vivo. Moreover, regardless of the parasite challenge, association of LaAg with MPL/AddaVax® did not significantly enhance protection in comparison with LaAg alone. This work demonstrated that MPL/AddaVax® is not effective in improving the efficacy of i.m. LaAg vaccine against cutaneous leishmaniasis.

The structure of the human cell cycle

ABSTRACTThe human cell cycle is conventionally depicted as a five-phase model consisting of four proliferative phases (G1, S, G2, M) and a single state of arrest (G0). However, recent studies show that individual cells can take different paths through the cell cycle and exit into distinct arrest states, thus necessitating an update to the canonical model. We combined time lapse microscopy, highly multiplexed single cell imaging and manifold learning to determine the underlying “structure” of the human cell cycle under multiple growth and arrest conditions. By visualizing the cell cycle as a complete biological process, we identified multiple points of divergence from the proliferative cell cycle into distinct states of arrest, revealing multiple mechanisms of cell cycle exit and re-entry and the molecular routes to senescence, endoreduplication and polyploidy. These findings enable the visualization and comparison of alternative cell cycles in development and disease.One-sentence summaryA systems-level view of single-cell states reveals the underlying architecture of the human cell cycle

MYC in Brain Development and Cancer

The MYC family of transcriptional regulators play significant roles in animal development, including the renewal and maintenance of stem cells. Not surprisingly, given MYC’s capacity to promote programs of proliferative cell growth, MYC is frequently upregulated in cancer. Although members of the MYC family are upregulated in nervous system tumours, the mechanisms of how elevated MYC promotes stem cell-driven brain cancers is unknown. If we are to determine how increased MYC might contribute to brain cancer progression, we will require a more complete understanding of MYC’s roles during normal brain development. Here, we evaluate evidence for MYC family functions in neural stem cell fate and brain development, with a view to better understand mechanisms of MYC-driven neural malignancies.