NNT-AS1 Impairs CD4+ T Cells Inltration by UpRegulating TGF-β Signaling in Hepatocellular Carcinoma

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 identied as one of negative factor for excessive immunological response. We sought to investigate the effects of NNT-AS1 mediating T cells inltration 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 signicant 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 signicantly lower than patients with low levels of NNT-AS1 (P=0.0402). Mechanistic analysis demonstrated that the downregulation of NNT-AS1 signicantly reduced the expression of TGF-β, TGFBR1, and SMAD5. Moreover, the inhibition of NNT-AS1 decrease the expression of TGF-β(cid:0)TGFBR1, and SMAD5. Importantly, IHC analyses indicated that the levels of NNT-AS1 were negatively correlated with CD4 + T lymphocyte cells inltration 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 inltration in HCC.

IHC analyses indicated that the levels of NNT-AS1 were negatively correlated with CD4 + T lymphocyte cells in ltration 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 in ltration in HCC.

Background
Hepatocellular carcinoma (HCC), as the dominant cause of death among liver cancer, is the 6th most common cancer in terms of incidence and has the 4th highest cancer-related mortality around the world [1]. In China, HCC morbidity and mortality rates rank as the 4th and 3rd in all malignant tumors respectively. Recently, immune checkpoint blockade (ICB), particularly antibody that targets PD-1, PD-L1, or CTLA-4, has been applied to treat HCC patients [2]. Nivolumab, pembrolizumab (PD-1 inhibitor) and tremelimumab (CTLA-4 inhibitor) have been demonstrated to be safe and effective in clinical trials [3][4][5]. Nivolumab has been approved to be as a second-line treatment for HCC [6]. In a phase I/II trial in advanced HCC (Checkmate-040 trial), nivolumab yielded a response rate of 20% [3]. Many factors contribute to prevent ICB therapy in HCC. Tumor in ltration lymphoccyes (TIL) is one of key factors associated with ICB therapy effect. For example, high in ltration of Foxp3 + T-cells was associated with poor prognosis [7]. However, the molecular mechanisms governing the establishment of TIL still remained to be illustrated in HCC.
LncRNAs are a subgroup of non-coding RNAs that are composed of more than 200 nt, which are involved in almost all key aspects of the occurrence, development and process of different types of tumors [8]. Many newly discovered lncRNAs have been reported to play as the oncogene or tumor suppressor role in a variety of tumorigenesis. LncRNA-ATB promotes metastasis of HCC [9]. HULC-mediated down-regulation of tumor suppressor p18 can promote cell proliferation in HCC [10]. LncRNA CUDR promotes the proliferation of HCC stem cells and affects the prognosis [11]. SNHG6-003 was found to function as a competing endogenous RNA to promote the progression and predict the poor prognosis of HCC [12]. NNT-AS1 is a novel identi ed lncRNA, which is located at 5p12 with 3 exons [13]. NNT-AS1 acts as one driver gene in cervical cancer [14], gastric cancer [15] and osteosarcoma [16]. NNT-AS1 was proved to induce Epithelial-to-Mesenchymal Transition (EMT) process in breast cancer by sponging miR-142-3p and up-regulating ZEB1 [17]. Downregulation of NNT-AS1 inhibits NSCLC progression via regulating miR-129-5p axis [18]. So far, the relationship between NNT-AS1 and TGF-β has not been studied. TGF-β signaling pathway plays key roles in regulating tumor immune reaction in Tumor Micro-Environment (TME) [19]. Tumor-derived TGF-β can induce tumorigenic and pro-metastatic responses in cancer cells and stroma, including the formation of an immune-suppressive TME [20]. Our previously data had proved that TGF-β signaling is hyper active in colon cancer. The loss of SMAD1/5/9 and TGFBR2 predicted a poor prognosis for colon cancer patients [21]. TGFβ consists of three isoforms (TGFB1, ~ 2, and ~ 3). TGFB1 is the most abundant and well-studied isoform [22]. There are two typical TGF-β receptors, TGF-β type I and type II receptors (TGFBR1 and TGFBR2), with active serine/threonine kinase [23]. Moreover, we demonstrated that the activation of TGF-β signaling could promote tumor angiogenesis, while the excessive activation of TGF-β signaling could prevent tumor angiogenesis [24]. TGF-β signaling mediates immune evasion by upregulating IDO in plasmacytoid DCs and CCL22 chemokine in myeloid DCs [25]. A recent study has provided that TGF-β induced CD8 + CD103 + cells present in tumor beds exhibiting a tolerogenic phenotype that facilitates immune evasion [26]. An antibody against αvβ8 integrin that blocks the release of active TGF-β by cancer cells unleashed the immune system against tumors in pre-clinical models, and this therapeutic effect was largely potentiated by combining anti-PD1 antibodies [27].
Here, we studied the levels of NNT-AS1 in HCC tissues by RNAscope in situ hybridization. Further analysis focused on the correlation between NNT-AS1 and the prognosis of patients with HCC. We discussed the mechanism that NNT-AS1 promoted TGF-β signaling activation in HCC cell lines. We also explored the correlation among NNT-AS1, TGF-β signaling, and TIL cells in the tissue samples from HCC.

Statistical analysis
The chi-squared tests and Cox proportional hazards regression model were performed using SPSS 11.0 statistical software. Chi-squared tests were applied where appropriate to analyze the differential expression of NNT-AS1 between cancer and normal tissues and its associations with clinicopathological parameters. GraphPad Prism 5 software was used to plot the OS rates. The signi cance of the FPKM values from TCGA datasets was determined using the Mann-Whitney U test. P < 0.05 was considered to be signi cant.

Results
High levels of NNT-AS1 are observed by RNAscope In Situ Hybridization in HCC 16 HCC patients were enrolled to compare the levels of NNT-AS1 between cancerous tissues and corresponding adjacent noncancerous tissues. A total of 32 tissue samples were successfully stained with NNT-AS1 by RNAscope (Fig. 1A, B). The scores were classi ed as follows: "-, +" represented low levels, and "++, +++, ++++" represented high levels of NNT-AS1. NNT-AS1 levels were demonstrated to be signi cantly increased in 10 (62.5%) of 16 HCC tissues. However, the NNT-AS1 molecule was rarely detected in corresponding adjacent noncancerous tissues; no (0%) tissue was ranked as having a high expression of NNT-AS1 (P = 0.001) ( Table 1).  [b]. Twenty-eight subjects lost.
High levels of NNT-AS1 are associated with shorter OS time in HCC tissues NNT-AS1 expression was further examined in HCC tissues by qRT-PCR cDNA. The samples included both cancerous tissues and adjacent noncancerous tissues that were derived from 64 HCC patients. Signi cantly higher NNT-AS1 expression was found in the cancerous tissues than that in the noncancerous tissues (P = 0.0003) ( Fig. 2A). Paired t test analysis was applied to compare the differential levels of NNT-AS1 in 26 paired cDNA samples. it was further con rmed that the levels of NNT-AS1 in cancer tissues were signi cantly higher than their levels that in adjacent non-cancerous tissues (Fig. 2B). Kaplan-Meier or Cox proportional hazards regression analyses were used to determine the prognostic relevant. In total, 64 subjects with HCCs were followed up for 2-113 months (mean ± S.D., 44.25 ± 31.45 months). 39 (60.94%) patients had died at the endpoint of follow up. Kaplan-Meier analysis indicated that the higher levels of NNT-AS1 predicated a shorter overall survival (OS) time than HCC patients to whom shown low levels of NNT-AS1 (P = 0.0402) (Fig. 2C). By univariate analysis (Cox proportional hazards regression), NNT-AS1 expression level (Higher vs. Lower, P = 0.044), tumor size ( < = 5 cm vs. >5 cm, P = 0.028), TNM stage (I vs. II/III, P = 0.036) were identi ed as the prognostic factors (Table 3). To further validate our ndings, we explored the difference in the expression levels of NNT-AS1 in the TCGA data. One of the HCC (LIHC) dataset that included 369 cancerous tissues and 50 normal tissues were used for the following investigations. We compared the normalized values (FPKM) that were determined by RNA sequencing of the cancerous tissues and normal tissues. In accordance with our results, the levels of NNT-AS1 were found to be signi cantly increased in HCC tissues when compared with NNT-AS1 levels in normal tissues (P < 0.0001) (Fig. 2D).
Follow-up information was available for 146 liver cancer patients from the LIHC dataset. Kaplan-Meier analysis demonstrated that higher NNT-AS1 FPKM values were signi cantly associated with reduced OS time compared with the corresponding low expression groups (P = 0.016) (Fig. 2E).  [29]. To investigate the roles for NNT-AS1 in HCC, we compared their levels in different immune types of HCC. NNT-AS1 were found to be signi cantly increased in C1 type ( Fig. 3A). C1 patients showed less tumor TILs and had shorter OS time [29]. We thus speculated that NNT-AS1 may regulate immune response in HCC.

Discussion
Increasing evidence has indicated that lncRNAs participate in a wide range of cellular processes, including regulation of epigenetic signatures, gene expression and proliferation [30,31] , [32] Since NGS detects the sequences in an unbiased way, NGS also enhances our knowledge about noncoding RNAs, such as long noncoding RNA (lncRNA), microRNA (miRNA) and circular RNA (circRNA), which were considered useless products of RNA splicing errors.Recently, Ye et al used NGS to nd that non-coding RNA was found to be involved in the regulation of many important physiological and pathological processes [33].Our present research demonstrated that NNT-AS1 is signi cantly increased and shows a robust correlation with HCC prognosis. In addition, we found that there was a positive correlation between the expression of NNT-AS1 and the activation of TGF-β signal. Importantly, the increased expression of NNT-AS1 inhibited CD4 + T cell tumor in ltration.
LncRNA was thought to be unstable and hard to preserve in para n-embedded tissues [32]. Here, we used the RNAscope method to evaluate the expression of NNT-AS1. This technique, which was rst developed by Wang et al., enabled the direct counting of mRNA molecules in single cells in routine formalin-xed tissue specimens using bright-eld microscopy [34]. More importantly, RNAscope has the advantage of avoiding false-negative results from the admixtures of many nonmalignant cells [35]. RNAscope analysis successfully captures RNA signals in para n les, thus accurate measurement of NNT-AS1 can be achieved. Through this advanced technology our study provided convincing evidence that HCC tissues showed signi cant increases in expression of NNT-AS1.
More and more lncRNAs have been reported to be involved in HCC immnue reaction. reported and predicted a poor prognosis of colon cancer [38]. Moreover, the signi cance of NNT-AS1 in orchestrating tumourigenesis had been addressed by many other reports. NNT-AS1 promoted cholangiocarcinoma cells proliferation and EMT through down-regulating miR-203 [39]. NNT-AS1 regulated the progression of lung cancer through the NNT-AS1/miR-3666/E2F2 axis [40]. NNT-AS1 promoted gastric cancer proliferation and invasion by regulating microRNA-363 expression [15]. Here, we conducted an analysis to address the novel mechanistic function of NNT-AS1 in HCC. Our analyses demonstrated that the expression of NNT-AS1 was positively associated with TGF-β signaling. TGF-β acts as immunosuppressive cytokine through effects on both immune cell differentiation and proliferation [41]. Collectively, we delineated NNT-AS1 as a prognostic factor with signi cantly increased levels in HCC. In addition, NNT-AS1 was proven to act as one of positive regulators of TGF-β, TGFBR1 and SMAD5. Further, NNT-AS1 was negatively associated with CD4 + cell TIL in HCC. Therefore, we demonstrated the novel mechanism that NNT-AS1 activated TGF-β signaling and further impaired CD4 + cell TIL. However, several limitations were present in this study. For instance, a retrospective approach was used to analyze the relationships between NNT-AS1 and clinical characteristics, and a relatively small cohort was enrolled. Further studies of the complete molecular mechanisms underlying NNT-AS1 knockdown would be useful to validate and expand our ndings.

Conclusion
Our study depicts a novel mechanism that conducts the process of CD4 + T lymphocyte cells in ltration. Of which is regulated by NNT-AS1 that activates TGF-β signaling pathway and thus impairs the CD4 + T lymphocyte cells in ltration in HCC. Our investigations suggest that the upregulation of NNT-AS1 serves as a promising predictor and might be conducive for clinicians to estimate OS time.

Declarations
Ethics approval and consent to participate    The levels of NNT-AS1 in different immune types of HCC(A). Pearson correlation analyzed the levels of NNT-AS1 and the genes that involved in TGF-β signaling and other genes as controls that involved in interferon signaling, angiogenesis, immunoreaction (B). qRT-PCR analysis of the relative mRNA levels of NNT-AS1 in three cell lines (HL-7702, HepG2,Huh7) (C). NNT-AS1 expression after transfecting siRNA that targeting TGFβ, TGFBR1, and SMAD5 was con rmed by qRT-PCR in HepG2 (D). hTGF-β activated the TGF-β signaling pathway and increased the expression of NNT-AS1. SB431542 inhibited the TGF-β signaling pathway and Page 20/21 decreased expression of NNT-AS1 (E, F). The correlation between NNT-AS1 with SMAD5 and TGFBR1 was analyzed by qRT-PCR in 15 HCC patients (G).