While the prognostic role of miRNAs in HCC has been reported in several studies [34], their role in the modulation of the TME remains largely unclear. In this study, we identified Tregs as a prognostic predictor for HCC using the CIBERSORT deconvolution machine learning algorithm, and identified 19 immune-related miRNAs significantly associated with the infiltration of Tregs in the TME. Two hub miRNAs showed significant prognostic performance in both the training and the validation datasets. These two miRNAs (hsa-miR-877 and hsa-miR-137) could, therefore, potentially serve as indicators for the infiltration of Tregs into the TME and predictors for the prognosis and immunotherapy responses of HCC patients under various clinical settings, including microenvironment cells, immune checkpoint markers, and antigen-presenting cells. Our analyses provide novel references for using miRNAs as potential candidate agents in ICI immunotherapy.
Tregs, a subset of CD4+ T cells, are central regulators of tolerance and immunity, stabilizing the suppressive phenotype of the TME [34]. Previous studies reported that the infiltration of Tregs in the TME is indicative of poor OS in various cancer subtypes [35-37]. Using machine learning techniques, we found that the enrichment of Tregs in the TME is one of the top predictors of poor prognosis in HCC patients. Consistent with our finding, Hsu P et al. reported that Tregs are major components of the immunosuppressive TME [38]. Moreover, another study showed that high levels of Tregs correlated with low responses in patients treated with ICIs [39]. This may be one of the main reasons for the correlation between high numbers of tumor-infiltrating Tregs and poor prognosis; in fact, the effect of Tregs on the microenvironment and immune tolerance may be key to poor outcomes.
Our studies highlight the fact that Tregs are regulated by more than Treg-intrinsic pathways, such as TGF-β activity and CCL22 [40], we found that Treg-related miRNAs also played a significant regulatory role. The targets of the 19 Treg-related miRNAs were significantly enriched in biological processes, including cellular responses to stress, mitotic cell cycle, myeloid cell differentiation and regulation of cellular response to stress, all of which have been implicated in the regulation of the differentiation of Tregs [41-43]. This finding further suggests a critical role for Treg-related miRNAs in Treg-induced pathogenesis. For example, miR15a/16 has been reported to directly target the 3’UTR regions of FOXP3, thus regulating Tregs at the post-transcriptional level [44]. Xia Lin et al. reported that miR-155-deficient mice exhibited a decrease in the numbers of Tregs in both the thymus and the spleen, and that miR-155 specifically targeted suppressor of cytokine signaling 1 (SOCS1), favoring Treg homeostasis [45]. Moreover, miR-146a-deficient Tregs were unable to suppress effector T cell activation [46]. Hence, miRNAs play an important role in regulating the differentiation and function of Tregs. The discovery of Treg-related miRNAs provides new insights into the underlying regulatory mechanisms of Tregs that result in the alteration of the TME and contribute to tumor biology and the therapeutic response to ICI.
We further examined the main hub Treg-related miRNAs (hsa-miR-877 and hsa-miR-137), which we showed were independent indicators of poor outcome. Hsa-miR-877 was found to be overexpressed in gastric cancer, and also antagonized the promoting effect of Substance P in the occurrence and progression of gastric cancer, suggesting that miR-877-5p could be a potential drug target [47]. Further, miR-877-5 has been reported to participate in gene regulation of competing endogenous RNA (ceRNA) mechanisms in multiple tumors, including cervical cancer, malignant gliomas, and non-small-cell lung cancer [48-50]. Yijun Yang et al. showed that Hsa-miR-137 effectively induced apoptosis and diminished tumorigenicity in multiple myeloma [51]. Chonglin Luo et al. also reported miR-137 could inhibit the invasion of melanoma cells [52]. Another report also suggested the prognostic relevance of miR-137 in patients with hepatocellular carcinoma [53]. We found that patients with high expression of either hsa-miR-877 or hsa-miR-137 had poorer prognosis than those with low expression of either hsa-miR-877 or hsa-miR-137. This shows that these hub Treg-related miRNAs (hsa-miR-877 and hsa-miR-137) are important prognostic markers for HCC patients.
It is widely accepted that the immune system homeostasis plays a key role in controlling tumor growth and metastasis. Multiple factors have been reported to affect escape from host’s immune system and resistance to anti-PD-1 monotherapy. These factors include lack of PD-L1 expression (TIL+/PD-L1−) [54], lack of cytotoxic T lymphocyte infiltration [55] and defects in antigen presenting cells [56]. To investigate the role of hsa-miR-877 and hsa-miR-137 in the host immune system, we evaluated the expression levels of TILs, HLA class I APM component and checkpoint markers, and found significant correlations between them. The results suggest that the regulation of tumor immunity by hsa-miR-877 and hsa-miR-137 may occur not only through Tregs, but also through the dysregulated expression of the APM component and by affecting the enrichment of TILs. Moreover, miRNAs have been found to be expressed in specific cells and tissues at special developmental stages and conditions. These facts suggest that the specific expression of hsa-miR-877 and hsa-miR-137 plays a key role in the fight against tumor cells by the host adaptive immune system. Further, we identified potential effects of hsa-miR-877 and hsa-miR-137 on the tumor immune microenvironment (TIM) of HCC, which suggests that hsa-miR-877 and hsa-miR-137 may be potential predictive biomarkers for the cancer immunotherapy response. As the immunosuppressive microenvironment is a major obstacle for successful tumor immunotherapy, the implementation of “combination immunotherapy” is a new hope for liver cancer treatment.
This study had some limitations. First, although the association between miRNA and Tregs could successfully predict HCC patients’ prognosis, there was only limited information on the clinical characteristics of the patient cohorts that we investigated. In addition, although a favorable performance in the external validation suggested that hsa-miR-877 and hsa-miR-137 could be potential candidates as regulators of the TIM, it is too early to conclude that immune-related miRNAs could act as novel immunotherapy reagents to reverse resistance in ICI non-responders in HCC. Therefore, further studies are required to confirm our findings.
This study aimed to provide new insights into the potential mechanisms involved in the role of miRNAs in immune escape and immune microenvironment regulation in HCC. Our data showed that hsa-miR-877 and hsa-miR-137 are key immune-related miRNAs that could affect the infiltration of Tregs in the TME. These data provide vital information that could help to guide further studies exploring potential combination immunotherapeutic approaches for HCC.