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

Yakun Wang,Xin Yang,Lei Yang,Zhe Liu,Tao Wen,Xichen Dong,Xin Zhao,Xinxue Zhang

doi:10.1155/2020/8216541

Abstract

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.

Highlights

Hepatocellular carcinoma (HCC) is a malignant disease with high mortality worldwide [1]
Generation sequence (NGS) has been widely used to explore novel differential long noncoding RNA (lncRNA) in cancer [26]. These lncRNAs participate in multiple tumorigenic processes and promote cancer cell proliferation and metastasis, [27]
Our present research identified a novel mechanism affecting tumor-infiltrating lymphocytes (TILs) in which nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) increased the activity of TGF-β signaling and inhibited CD4 T cell infiltration

Summary

Introduction

Hepatocellular carcinoma (HCC) is a malignant disease with high mortality worldwide [1]. Increasing tumor-infiltrating lymphocyte (TIL) levels is the most important way to improve ICB treatment [5]. P18 is a cancer suppressor, whose expression can be downregulated by lncRNA/HULC to increase HCC cell proliferation [9]. Tumor immune evasion can be mediated by TGF-β signaling that increases IDO and CCL22 expressions in dendritic cells (DCs) [16]. A new ICB treatment strategy employing antibodies that could block both TGF-β and PD1 was developed. Those antibodies have been validated in a preclinical model [18]. Our mechanistic analyses uncovered the novel mechanism by which NNT-AS1 activates TGF-β signaling and the associated effects on TILs in HCC

Materials and Methods

Results

Discussion

Conflicts of Interest