LncRNA MAGI2-AS3 Is Regulated by BRD4 and Promotes Gastric Cancer Progression via Maintaining ZEB1 Overexpression by Sponging miR-141/200a

Abstract

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and tumor progression. However, the biological function of most lncRNAs remains unknown in human gastric cancer. This study here aims to explore the unknown function of lncRNA MAGI2-AS3 in gastric cancer. First, bioinformatics analysis showed that lncRNA MAGI2-AS3 was overexpressed in gastric cancer tissues, and the overexpression of MAGI2-AS3 has been shown to be associated with poor prognosis in all three independent gastric cancer cohorts (The Cancer Genome Atlas stomach cancer [TCGA_STAD], GEO: GSE62254 and GSE15459). The multivariate analysis indicated that lncRNA MAGI2-AS3 was an independent prognostic factor for both overall survival and disease-free survival of gastric cancer patients. Moreover, MAGI2-AS3 was identified to be an epithelial-mesenchymal transition (EMT)-related lncRNA and was highly co-expressed with ZEB1/2 in both gastric cancer tissues and normal stomach tissues. Loss-of-function and gain-of-function studies showed that lncRNA MAGI2-AS3 could positively regulate ZEB1 expression and the process of cell migration and invasion in gastric cancer. Subcellular location assay showed that lncRNA MAGI2-AS3 was mainly located in the cytoplasm of gastric cancer cells. Bioinformatics analysis and functional experiments revealed that lncRNA MAGI2-AS3 was negatively correlated with miR-141/200a expression and negatively regulated miR-141/200a-3p expression in gastric cancer. Therefore, we speculate that lncRNA MAGI2-AS3 promotes tumor progression through sponging miR-141/200a and maintaining overexpression of ZEB1 in gastric cancer. Nevertheless, we identified that BRD4 is a transcriptional regulator of lncRNA MAGI2-AS3 in gastric cancer. Additionally, our findings highlight that lncRNA MAGI2-AS3 is an ideal biomarker and could be a potential therapeutic target for gastric cancer.

Keywords: BRD4; ZEB1; gastric cancer; lncRNA MAGI2-AS3; miR-141; miR-200a.

Figure 1

The Clinical Significance of lncRNA MAGI2-AS3 Expression Was Analyzed in TCGA-STAD Cohort (A) Difference in expression levels of MAGI2-AS3 between GSE79973 and GSE54129 stomach cancer cohorts. (B) Difference in expression levels of MAGI2-AS3 between intestinal gastric cancer tissues and diffuse gastric cancer tissues. (C) Difference in expression levels of MAGI2-AS3 in gastric cancer tissues with different degrees of differentiation. (D–G) MAGI2-AS3 expression level in different T stages (D), pathologic stages (E), N stages (F), and M stages (G) of gastric cancer. (H and I) Kaplan-Meier analysis of overall survival time (H) and disease-free time (I) of patients with gastric cancer according to the expression of lncRNA MAGI2-AS3. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 2

The Clinical Significance of lncRNA MAGI2-AS3 Expression Was Analyzed in the GSE62254 and GSE15459 Cohorts (A) Difference in expression levels of MAGI2-AS3 between intestinal gastric cancer tissues and diffuse gastric cancer tissues. (B–E) MAGI2-AS3 expression level in different T stages (B), pathologic stages (C), N stages (D), and M stages (E) of gastric cancer tissues. (F and G) Kaplan-Meier analysis of overall survival time (F) and disease-free time (G) of gastric cancer patients in the GES62254 cohort according to the expression of lncRNA MAGI2-AS3. (H and I) Kaplan-Meier analysis of overall survival time (H) and disease-free survival time (I) in the gastric cancer cohort of GSE15459 according to the expression of lncRNA MAGI2-AS3. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 3

MAGI2-AS3 Was an EMT-Related lncRNA and Highly Co-expressed with ZEB1/2 in Normal Gastric Tissues and Gastric Cancer Tissues (A) Expression level of lncRNA MAGI2-AS3 in the four subtypes (MSS/TP53⁻, MSS/TP53⁺, MSI, and EMT) of gastric cancer in the GSE62254 cohort. (B) R values of co-expression between different genes and lncRNA MAGI2-AS3 were analyzed in the GSE62254 cohort. (C and D) lncRNA MAGI2-AS3 was highly co-expressed with ZEB1 (C) and ZEB2 (D) in the GSE62254 cohort. (E) R values of co-expression between different genes and lncRNA MAGI2-AS3 were analyzed in the TCGA_STAD cohort. (F and G) lncRNA MAGI2-AS3 was highly co-expressed with ZEB1 (F) and ZEB2 (G) in TCGA-STAD cohort. (H and I) lncRNA MAGI2-AS3 was highly co-expressed with ZEB1 (H) and ZEB2 (I) in normal stomach tissues from the GTEx dataset. ****p < 0.0001.

Figure 4

MAGI2-AS3 Positively Regulated Gastric Cancer Cell Migration and Invasion, and Positively Regulated ZEB1 Expression in gastric cancer (A) lncRNA MAGI2-AS3 expression level in different gastric cancer cell lines was determined by qRT-PCR assay. (B) Relative lncRNA MAGI2-AS3 expression level in cytoplasm and nuclear of the HGC-27 cell line was determined by qRT-PCR assay. (C and D) The efficiency of knockdown (C) and overexpression (D) of lncRNA MAGI2-AS3 was determined by qRT-PCR assay. (E and F) The wound healing assays were performed in gastric cancer cells after knockdown (E) and overexpression (F) of lncRNA MAGI2-AS3. (G) The Transwell migration assays were performed in gastric cancer cells after knockdown and overexpression of lncRNA MAGI2-AS3. (H) The Transwell invasion assays were performed in gastric cancer cells after knockdown and overexpression of lncRNA MAGI2-AS3. (I and J) Relative ZEB1 expression level in gastric cancer cells after knockdown (I) and overexpression (J) of lncRNA MAGI2-AS3 was determined by qRT-PCR assay. **p < 0.01.

Figure 5

LncRNA MAGI2-AS3 Was Negatively Correlated with miR-141/200a Expression in gastric cancer Tissues (A) Possible miRNA families that could interact with lncRNA MAGI2-AS3 were predicted by two different online web tools. (B) The binding region and the folding energy of miR-141/200a family and MAGI2-AS3 were predicted by the RNA22 web server. (C) R values of co-expression between different miRNAs and lncRNA MAGI2-AS3 were analyzed in the TCGA_STAD cohort. (D and E) lncRNA MAGI2-AS3 showed a negative correlation with miR-141 (D) and miR-200a (E) expression in the TCGA_STAD cohort. (F) The difference in expression level of miR-141 and miR-200a between the high MAGI2-AS3 expression group and low MAGI2-AS3 expression group in the TCGA_STAD cohort. (G and H) The difference in expression level of ZEB1 between high MAGI2-AS3 expression group and low MAGI2-AS3 expression group in the TCGA_STAD cohort (G) and GSE62254 cohort (H). ****p < 0.0001.

Figure 6

LncRNA MAGI2-AS3 Negatively Regulated miR-141 and miR-200a Expression in gastric cancer (A and B) Difference in expression levels of miR-141 (A) and miR-200a (B) in different T stages of gastric cancer tissues in TCGA cohort. (C) Difference in expression levels of miR-141/200a in gastric cancer tissues with different degrees of differentiation. (D) Difference in expression levels of miR-141/200a between intestinal gastric cancer tissues and diffuse gastric cancer tissues from TCGA cohort. (E and F) Kaplan-Meier analysis of overall survival time in TCGA cohort according to the expression of miR-141 (E) and miR-200a (F). (G and H) Difference in expression levels of miR-141 (G) and miR-200a (H) in normal stomach tissues and different T stage of gastric cancer tissues from the GSE26595 cohort. (I and J) Relative miR-141 and miR-200a expression levels in gastric cancer cells after knockdown (I) and overexpression (J) of lncRNA MAGI2-AS3 was determined by qRT-PCR assay. ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 7

BRD4 Transcriptionally Regulated lncRNA MAGI2-AS3 Expression in gastric cancer (A) Abundant H3K27Ac marks were observed in the promoter region of MAGI2-AS3 according to the UCSC web server. (B) lncRNA MAGI2-AS3 expression remarkably decreased in the HGC-27 cells treated with 2.5 μM JQ1 compared with negative control. (C) Knockdown efficiency of BRD4 by RNA interference in HGC-27 cell line. (D and E) Knockdown of BRD4 expression reduced lncRNA MAGI2-AS3 expression (D) and ZEB1 expression (E) in gastric cancer. (F and G) Kaplan-Meier analysis of overall survival time in the gastric cancer cohort of GSE62254 according to BRD4 (F) and ZEB1 (G) expression levels. (H) Kaplan-Meier analysis of overall survival time in the gastric cancer cohort of TCGA according to ZEB1 expression. **p < 0.01.