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. 2022 Mar 3:12:789283.
doi: 10.3389/fonc.2022.789283. eCollection 2022.

Identification of N6-Methyladenosine-Related lncRNAs as a Prognostic Signature in Glioma

Yujia Chen  1 , Yuduo Guo  2 , Shenglun Li  1 , Jiacheng Xu  1 , Xiang Wang  1 , Weihai Ning  1 , Lixin Ma  1 , Yanming Qu  1 , Mingshan Zhang  1 , Hongwei Zhang  1
Affiliations

Identification of N6-Methyladenosine-Related lncRNAs as a Prognostic Signature in Glioma

Yujia Chen et al. Front Oncol. .

Abstract

N6-methyladenosine (m6A) modification is the most abundant modification in long noncoding RNAs (lncRNAs). Current studies have shown that the abnormal expression of m6A-related genes is closely associated with the tumorigenesis and progression of glioma. However, the role of m6A-related lncRNAs in glioma development is still unclear. Herein, we screened 566 m6A-related lncRNAs in glioma from The Cancer Genome Atlas (TCGA) database. The expression pattern of these lncRNAs could cluster samples into two groups, in which various classical tumor-related functions and the tumor immune microenvironment were significantly different. Subsequently, a nine-factor m6A-related lncRNA prognostic signature (MLPS) was constructed by using a LASSO regression analysis in the training set and was validated in the test set and independent datasets. The AUC values of the MLPS were 0.881, 0.918 and 0.887 for 1-, 3- and 5-year survival in the training set, respectively, and 0.856, 0.916 and 0.909 for 1-, 3-, and 5-year survival in the test set, respectively. Stratification analyses of the MLPS illustrated its prognostic performance in gliomas with different characteristics. Correlation analyses showed that the infiltrations of monocytes and tumor-associated macrophages (TAMs) were significantly relevant to the risk score in the MLPS. Moreover, we detected the expression of four MLPS factors with defined sequences in glioma and normal cells by using RT-PCR. Afterwards, we investigated the functions of LNCTAM34A (one of the MLPS factors) in glioma cells, which have rarely been reported. Via in vitro experiments, LNCTAM34A was demonstrated to promote the proliferation, migration and epithelial-mesenchymal transition (EMT) of glioma cells. Overall, our study revealed the critical role of m6A-related lncRNAs in glioma and elucidated that LNCTAM34A could promote glioma proliferation, migration and EMT.

Keywords: N6-methyladenosine; The Cancer Genome Atlas; glioma; long non-coding RNA; prognostic signature.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) The coexpression network of m6A regulators and m6A-related lncRNAs. (B) Consensus matrix of the consensus clustering based on the m6A-related lncRNAs for optimal k = 2. (C) Area under the CDF curve increment for k = 2 to 9. (D) The heatmap of 50 randomly m6A-related lncRNAs in 2 clusters. (E) Overall survival of patients (TCGA) in different clusters.
Figure 2
Figure 2
(A) The percentages of monocytes, resting memory CD4 T cells, activated mast cells, TAMs, immune scores and stromal scores of gliomas in the two clusters (****p < 0.0001, **p < 0.01). (B) Different functional pathway enrichments in the two clusters.
Figure 3
Figure 3
Flow chart of this study. DE, differentially expressed; Pro, promotor; Sup, suppressor. RT–PCR Quantitative Real-Time Polymerase Chain Reaction.
Figure 4
Figure 4
(A) The volcano plot of m6A-related lncRNAs in glioma. (B) Heatmap of the 100 top differentially expressed m6A-related lncRNAs. (C) The 144 key m6A-related lncRNAs. (D-E) LASSO Cox regression analysis of m6A-related lncRNAs.
Figure 5
Figure 5
(A) The distributions of risk scores, alive/dead status and the expression of nine m6A-related lncRNAs in the training set. (B) Overall survival analysis for patients in the high- and mid-/low-risk groups. (C) Univariate and (D) multivariate Cox regression analyses for OS in glioma patients in the training set. (E) ROC curve of the risk score at different follow-up times. (F) The ROC curve of the risk score and other clinical characteristics.
Figure 6
Figure 6
(A) The distributions of risk scores, alive/dead status and the expression of nine m6A-related lncRNAs in the test set. (B) Overall survival analysis for patients in the high- and mid-/low-risk groups. (C) Univariate Cox regression analyses and (D) multivariate Cox regression analyses for OS in glioma patients in the test set. (E) ROC curve of the risk score at different follow-up times. (F) The ROC curve of the risk score and other clinical characteristics.
Figure 7
Figure 7
(A) Heatmap of MLPS and clinicopathological features. (B) Differences in stromal scores and immune scores in the high- and low-risk groups. (C) The correlation of immune cells and risk score. MLPS, m6A-related lncRNA prognostic signature; ****p < 0.0001.
Figure 8
Figure 8
Knockdown of LNCTAM34A reduced proliferation, migration and EMT in glioma cells. (A) The expression levels of CRNDE, LINC00641 and LNCTAM34a in glioma cell lines (U87-MG, LN229 and U343) and normal human astrocytes (SVGP12) as detected by RT–PCR. (B) LNCTAM34A expression levels in U87-MG cells or LN229 cells transfected with siNC or two different specific siRNAs. The CCK-8 assay was used to detect (C) U87-MG cell or (E) LN229 cell proliferation at 0, 12, 36 and 48 h after incubation.The Transwell assay was used to detect (D) U87-MG cells or (F) LN229 cell migration at 24 h after incubation. (G) The expression of E-cadherin and Vimentin in U87-MG cells and (H) LN229 cells was measured by using Western blotting. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
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