Long Non-Coding RNA LOC339059 Attenuates IL-6/STAT3-Signaling-Mediated PDL1 Expression and Macrophage M2 Polarization by Interacting with c-Myc in Gastric Cancer

Abstract

Background: Long non-coding RNA (lncRNA) was identified as a novel diagnostic biomarker in gastric cancer (GC). However, the functions of lncRNAs in immuno-microenvironments have not been comprehensively explored. In this study, we explored a critical lncRNA, LOC339059, that can predict the clinical prognosis in GC related to the modulation of PD-L1 and determined its influence upon macrophage polarization via the IL-6/STAT3 pathway. Methods: To date, accumulating evidence has demonstrated that the dysregulation of LOC339059 plays an important role in the pathological processes of GC. It acts as a tumor suppressor, regulating GC cell proliferation, migration, invasion, tumorigenesis, and metastasis. A flow cytometry assay showed that the loss of LOC339059 enhanced PDL1 expression and M2 macrophage polarization. RNA sequencing, RNA pull-down, RNA immunoprecipitation, Chip-PCR, and a luciferase reporter assay revealed the pivotal role of signaling alternation between LOC339059 and c-Myc. Results: A lower level of LOC339059 RNA was found in primary GC tissues compared to adjacent tissues, and such a lower level is associated with a poorer survival period (2.5 years) after surgery in patient cohorts. Moreover, we determined important immunological molecular biomarkers. We found that LOC339059 expression was correlated with PD-L1, CTLA4, CD206, and CD204, but not with TIM3, FOXP3, CD3, C33, CD64, or CD80, in a total of 146 GC RNA samples. The gain of LOC339059 in SGC7901 and AGS inhibited biological characteristics of malignancy, such as proliferation, migration, invasion, tumorigenesis, and metastasis. Furthermore, our data gathered following the co-culture of THP-1 and U937 with genomic GC cells indicate that LOC339059 led to a reduction in the macrophage cell ratio, in terms of CD68+/CD206+, to 1/6, whereas the selective knockdown of LOC339059 promoted the abovementioned malignant cell phenotypes, suggesting that it has a tumor-suppressing role in GC. RNA-Seq analyses showed that the gain of LOC339059 repressed the expression of the interleukin family, especially IL-6/STAT3 signaling. The rescue of IL-6 in LOC339059-overexpressing cells reverted the inhibitory effects of the gain of LOC339059 on malignant cell phenotypes. Our experiments verified that the interaction between LOC339059 and c-Myc resulted in less c-Myc binding to the IL-6 promoter, leading to the inactivation of IL-6 transcription. Conclusions: Our results establish that LOC339059 acts as a tumor suppressor in GC by competitively inhibiting c-Myc, resulting in diminished IL-6/STAT3-signaling-mediated PDL1 expression and macrophage M2 polarization.