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Background Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) is one of long non coding RNA, has been shown with high levels in several types of cancers. However, the molecular mechanism remains to be revealed for NNT-AS1 in the progession of hepatocellular carcinoma (HCC). TGF-β signaling pathway has been identified as one of negative factor for excessive immunological response. We sought to investigate the effects of NNT-AS1 mediating T cells infiltration by regulating TGF-β signaling pathway in patients with HCC.
Methods RNAscope In Situ Hybridization and Real Time Quantitative PCR assays were applied to detect NNT-AS1 levels in HCC tissues. Immunohistochemistry (IHC) assays were used to observe the co-expressions of TGF-β, TGFBR1, SMAD1/5/9, and CD4+ T cells. The mechanisms as to how NNT-AS1 orchestrates TGF-β signaling were further explored in HepG2 and Huh7 cells.
Results RNA-scope analyses revealed that the levels of NNT-AS1 were significant higher in cancerous tissues than in paired non-cancerous tissues (P=0.0001). Quantitative PCR assays validated the high expression of NNT-AS1 in HCC cancer tissues (n=64) when compared with normal tissues (n=26) (P=0.0003). The prognostic analysis indicated that the OS rates for HCC patients with high levels of NNT-AS1 were significantly lower than patients with low levels of NNT-AS1 (P=0.0402). Mechanistic analysis demonstrated that the downregulation of NNT-AS1 significantly reduced the expression of TGF-β, TGFBR1, and SMAD5. Moreover, the inhibition of NNT-AS1 decrease the expression of TGF-β，TGFBR1, and SMAD5. Importantly, IHC analyses indicated that the levels of NNT-AS1 were negatively correlated with CD4+ T lymphocyte cells infiltration in tissues from 16 HCC patients.
Conclusions Our study depicts a novel mechanism regarding NNT-AS1 activates TGF-β signaling pathway and thus impairs the CD4+ T lymphocyte cells infiltration in HCC.
Keywords
Hepatocellular Carcinoma (HCC), long noncoding RNA, NNT-AS1, TGF-β, CD4+ T cell
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Primary research
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Complete author information
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CURRENT STATUS: Posted
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Posted 22 Jun, 2020
No community comments so far
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Subject Areas
Cancer Biology
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This work is licensed under a CC BY 4.0 License. Read Full License
Background Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) is one of long non coding RNA, has been shown with high levels in several types of cancers. However, the molecular mechanism remains to be revealed for NNT-AS1 in the progession of hepatocellular carcinoma (HCC). TGF-β signaling pathway has been identified as one of negative factor for excessive immunological response. We sought to investigate the effects of NNT-AS1 mediating T cells infiltration by regulating TGF-β signaling pathway in patients with HCC.
Methods RNAscope In Situ Hybridization and Real Time Quantitative PCR assays were applied to detect NNT-AS1 levels in HCC tissues. Immunohistochemistry (IHC) assays were used to observe the co-expressions of TGF-β, TGFBR1, SMAD1/5/9, and CD4+ T cells. The mechanisms as to how NNT-AS1 orchestrates TGF-β signaling were further explored in HepG2 and Huh7 cells.
Results RNA-scope analyses revealed that the levels of NNT-AS1 were significant higher in cancerous tissues than in paired non-cancerous tissues (P=0.0001). Quantitative PCR assays validated the high expression of NNT-AS1 in HCC cancer tissues (n=64) when compared with normal tissues (n=26) (P=0.0003). The prognostic analysis indicated that the OS rates for HCC patients with high levels of NNT-AS1 were significantly lower than patients with low levels of NNT-AS1 (P=0.0402). Mechanistic analysis demonstrated that the downregulation of NNT-AS1 significantly reduced the expression of TGF-β, TGFBR1, and SMAD5. Moreover, the inhibition of NNT-AS1 decrease the expression of TGF-β，TGFBR1, and SMAD5. Importantly, IHC analyses indicated that the levels of NNT-AS1 were negatively correlated with CD4+ T lymphocyte cells infiltration in tissues from 16 HCC patients.
Conclusions Our study depicts a novel mechanism regarding NNT-AS1 activates TGF-β signaling pathway and thus impairs the CD4+ T lymphocyte cells infiltration in HCC.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
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