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DOI: 10.1055/s-0037-1612834
Increase HCC proliferation with TGF-βR1 inhibition and TLR7 stimulation; are they two faces for one coin?
Publication History
Publication Date:
03 January 2018 (online)
Background:
Around 70%-90% of hepatocellular carcinoma (HCC) cases are observed in patients with liver cirrhosis and long-standing hepatic inflammation. Our previous work showed that HCC proliferation can be modulated by toll-like receptor 7 and 9. Moreover, an association between TLR activity and TGF-β signaling was reported previously in liver fibrosis, suggesting that TGF-β can be incorporated in the process of HCC modulation through TLRs.
Aim of the work:
Our aim is to investigate the impact of TGF-βR1 and TLR7 pathways modulation on HCC proliferation in an in vivo mouse model and cell line. Further, we will determine whether inhibition of TGF-β pathway can modulate Toll-like receptor and in turn HCC proliferation.
Materials and methods:
Immunohistochemistry with the following antibodies TLR7, TGF-β1, pSmad2, and Ki-67 was performed on liver fibrosis and HCC tissue obtained from steatosis-based HCC mouse model. HuH7 cells were treated with imiquimod (5 µg/ml, TLR7 agonist), chloroquine (20µM/ml, inhibitor for TLR7), TGF-β1 cytokine (5 ng/ml) and LY2157299 (10 ng/ml, TGF-βR1 inhibitor) in order to detect the effect of TLR7 and TGF-βR1 modulation (stimulation and inhibition) on cellular proliferation by using MTS assay. Further, the expression level TLR7 upon TGF-βR1 stimulation and inhibition was evaluated on protein and mRNA levels and PCNA expression was measured by western blot.
Results:
The expressions of TLR7 and TGF-βR1 in all HCC nodules compared to non-tumoral livers were conversely correlated. Moreover, TLR7expression was positively correlated with the proliferative index measured by Ki-67. Moreover, high TGF-βR1 and pSmad2 expression in dysplastic nodules and fibrotic livers were observed, whereas, HCC nodules were not expressed. Localization of pSmad2 positive signals HCC was perinuclear more than nuclear. In HuH7 cells, proliferation was significantly associated with TLR7 stimulation (imiquimod) and TGF-βR1 inhibition (Ly2157299) using MTS assay. This result was confirmed by PCNA expression using western blot. Surprisingly, TLR7 expression was increased upon LY2157299 incubation on both mRNA and protein levels.
Conclusion:
Targeting the TGF-βR1 and TLR7 pathways can be used to modulate HCCproliferation.