Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/β-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells

Abstract

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/β-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of β-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/β-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the β-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/β-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

Chicken telomerase reverse transcriptase mediates LMH cell pyroptosis by regulating the nuclear factor-kappa B signaling pathway

“…Avian leghorn male hepatoma ( LMH ) cell line stably overexpressing the chTERT (HA-tag) gene (LMH-chTERT) and control cells (LMH-NC) were constructed by our laboratory in previous study ( Xiang et al, 2021 ). The cells were cultured in DMEM/F12 (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum and maintained at 37°C and 5% CO 2 .…”

“…LMH cells are an avian-derived tumor cell line, which can be used as an in vitro research model for tumor diseases such as avian leukosis and Marek's disease. Our previous research results showed that overexpression of chTERT can up-regulate the Wnt/β-catenin signaling pathway to shorten the LMH cell cycle, inhibit cell apoptosis, promote cell proliferation and the replication of avian leukosis virus subgroup J ( ALV-J ); conversely, the expression of chTERT can also be upregulated after infected ALV-J ( Xiang et al, 2021 ). This study showed that overexpression of chTERT can promote pyroptosis by up-regulating the NF-κB signaling pathway in LMH cells.…”

The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

“…The results showed that the methylation level of the chTERT promoter region in LMH cells was higher than that in DF-1 cells (Figure 2). Our previous study showed that telomerase activity was positive in LMH cells and negative in DF-1 cells [33]; that is, chTERT was not expressed in DF-1 cells. In conclusion, the expression of chTERT in LMH cells is related to the hypermethylation level of the promoter region.…”

“…Expression levels were quantified using the 2 −ΔΔCt method and normalized to GAPDH expression. The sequences of the primers used were the same as those used in our previous study [33].…”

Avian Leucosis Virus-Host Interaction: The Involvement of Host Factors in Viral Replication

“…The chicken telomerase reverse transcriptase (chTERT) might play a regulatory role in the process. chTERT is mutually regulated with the Wnt/β-catenin signaling pathway to inhibit apoptosis, promote ALV-J replication, and increase telomerase activity ( 71 ). The activation of the ERK/MAPK pathway is required for ALV-J replication and is associated with virus-induced tumorigenesis ( 72 ).…”