Glioma is a common nervous system cancer that represents difficulties in completely surgical removal. Along with the resistance to radiotherapy and chemotherapy, glioma has become one of the most malignant tumors[23, 24]. LGGs are infiltrative gliomas that most often occur in the adult cerebral hemispheres, a subset of LGGs stays stable for several years, whereas some progress into GBM within a few months, causing a dismal prognosis. Hence, understanding the molecular mechanisms of glioma initiation and progression is important in developing effective therapeutic strategies for LGG patients. In this study, we identified miR-200c, which belongs to the tumor suppressor miR-200 family, is crucial for IDH1-mutant LGG patients through activating an immune response.
MiR-200c belongs to the miR-200 family. In recent years, an increasing number of studies have shown that miR-200c expression inhibits the progression, invasion, EMT and chemoresistance of a variety of tumors, including non-small cell lung cancer, breast cancer stem cells, prostate cancer and ovarian cancer [26, 27] [28, 29]. Furthermore, miR-200c/141 were reported to inhibit the expression of transcription factors ZEB1 and ZEB2, which act as markers of EMT, resulting in the downregulation of E-cadherin expression and reduction of cell adhesion and finally promoting the invasion and metastasis of tumor cells[30, 31]. These results indicated that despite the mutually genetic alterations with IDH1 mutation existing in LGG patients, no miR-200c mutation was detected, which eventually resulted in better overall survival. It is consistent with our results, that is, compared with IDH1 wild-type tumors, IDH1-mutant tumors with a relatively higher ZEB1/2 expression showed a lower expression of miR-200c/141, indicating the vital role of miR-200c as a tumor suppressor through regulating the expression of ZEB1/2. The opposite expression pattern of miR-200c/141 and ZEB1/2 demonstrates the negative regulatory feedback loop between miR-200 family members and ZEB TFs, future studies will be needed to test in LGG samples.
IDH1 mutations are closely related to epigenetic variations. Epigenetic mechanisms, especially DNA methylation, play a decisive role during tumorigenesis and EMT and contribute to the regulation of key factors involved in this process. The hypermethylation in regulatory regions of many tumor suppressor genes leads to their transcriptional silencing (e.g. E-cadherin). MiR-200c and miR-141 are both located on chromosome 12p13, however, Niu et al. identified a transcriptional unit containing miR-200c, but not miR-141, within the CpG Island at position -285. The result of an alternative splicing variant exists to splice out miR-141 and conserve the miR-200c transcript was supported by an in-silico inspection, upon which a high-score splice donor site (boundary exon/intron) was detected between the two miRNAs in position +373. This data might explain the observation previously made by us and others[20, 21], that miR-200c is usually expressed at higher levels and is more affected by methylation level and copy number variation than miR-141, despite their close location.
Hypermethylation of CpG islands in promoter regions contributes to the miR-200c expression. According to the epigenetic methylation level, IDH1-mutant LGGs are normally classified as methylation-rich. In our case, the methylation level of the miR-200c gene was detected by the six probes on the CpG island before the transcription start site of miR-200c. Moreover, there is a significant association between methylation level and amplification-type alteration, which were both associated with the expression level of miR-200c. To further understand the molecular mechanism behind the effect of miR-200c in IDH1-mutant LGG tissues, we performed RNA-seq analysis, downstream GO analysis, and GSEA. The results illustrated that miR-200c might be an important regulator of the immune response, which was consistent with the previous study that miR-200c can control the inflammatory process and the response to an infection[33–35].
It was shown that miR-200c has the ability to modify the efficiency of TLR4 signaling through the MyD88-dependent pathway, thus influencing host innate defenses against microbial pathogens. Rogers, T.J., et al. illustrated that miR-200c led to reduced production of the immune-suppressive metabolite kynurenine directly by targeting TDO2. Furthermore, in addition to reversing a classical EMT signature, miR-200c represses many genes encoding immune-suppressive factors containing PD-L1/2, HMOX1, and GDF15. It is also recognized that inflammatory-related miR-200c can sustain innate immune responses through LPS-dependent mechanisms, strengthening our result that miR-200c plays a vital role in immune responses, providing a therapeutic strategy via a miR-200c-modulated immune response.
These results were further confirmed by WGCNA and downstream enrichment analysis, showing that the genes in the yellow module played essential roles in immune responses. Besides, the genes highly correlated with miR-200c expression showed intensive connection by the visualization of metascape. A variety of the hub-genes were immune proteins, e.g., CD180, ITGAL, SYK, ITGB2, CD53, and LAPTM5, indicating the high association between miR-200c and immune systems.