Overexpression of NNT-AS1 Activates TGF-β Signaling to Decrease Tumor CD4 Lymphocyte Infiltration in Hepatocellular Carcinoma

Abstract EN

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC).

However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated.

The activation of TGF-β signaling inhibits tumor-infiltrating lymphocytes (TILs) and results in tumor immune evasion.

We thus planned to explore the mechanism by which NNT-AS1 activates the TGF-β signaling pathway and inhibits TILs in HCC.

High levels of NNT-AS1 were detected in HCC tissues by both RNAscope and real-time quantitative PCR (RT-qPCR) assays.

The levels of proteins involved in TGF-β signaling and those of CD4 T lymphocytes were quantified by immunohistochemistry (IHC).

HCC cell lines (HepG2 and Huh7) were used to explore the effects of NNT-AS1 on TGF-β signaling activation.

In these analyses, RNAscope detection demonstrated that NNT-AS1 levels were significantly increased in HCC cancer tissues (P=0.0001).

In addition, the elevated NNT-AS1 levels in cancer tissue were further confirmed by RT-qPCR analysis of HCC cancer tissues (n=64) and normal tissues (n=26) (P=0.0003).

Importantly, the overall survival time of HCC patients who exhibited higher levels of NNT-AS1 expression was significantly shorter than that of HCC patients who had lower levels of NNT-AS1 expression (P=0.0402).

Further mechanistic investigation indicated that NNT-AS1 inhibition significantly decreased the levels of TGF-β, TGFBR1, and SMAD5 in HCC cells.

In HCC tissues, IHC detection showed that relatively high NNT-AS1 levels were associated with a reduction in infiltrated CD4 lymphocyte numbers.

In conclusion, this research identifies a novel mechanism by which NNT-AS1 impairs CD4 T cell infiltration via activation of the TGF-β signaling pathway in HCC.