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. 2020 Oct 9:2020:6575724.
doi: 10.1155/2020/6575724. eCollection 2020.

lncRNA DLGAP1-AS2 Knockdown Inhibits Hepatocellular Carcinoma Cell Migration and Invasion by Regulating miR-154-5p Methylation

Kai Chen  1 , Zhuqing Zhang  2 , Aijun Yu  1 , Jian Li  1 , Jinlong Liu  1 , Xuejun Zhang  1
Affiliations

lncRNA DLGAP1-AS2 Knockdown Inhibits Hepatocellular Carcinoma Cell Migration and Invasion by Regulating miR-154-5p Methylation

Kai Chen et al. Biomed Res Int. .

Abstract

Objective: DLGAP1-AS2 has been characterized as an oncogenic lncRNA in glioma. Our preliminary microarray analysis revealed the altered expression of DLGAP1-AS2 in hepatocellular carcinoma (HCC), but the role of DLGAP1-AS2 in HCC remains unknown.

Method: Expression of DLGAP1-AS2 and miR-154-5p in paired HCC and nontumor tissues from 62 HCC patients was determined by RT-qPCR. The 62 HCC patients were followed up for 5 years to analyze the prognostic value of DLGAP1-AS2 for HCC. DLGAP1-AS2 knockdown and miR-154-5p overexpression was achieved in HCC cells to study the relationship between them. Methylation of miR-154-5p was analyzed by methylation-specific PCR. Cell proliferation was analyzed by CCK-8 assay.

Results: DLGAP1-AS2 was upregulated in HCC and predicted poor survival. miR-154-5p was downregulated in HCC and inversely correlated with DLGAP1-AS2. In HCC cells, DLGAP1-AS2 knockdown resulted in the upregulation of miR-154-5p expression and decreased methylation of miR-154-5p gene. Transwell assay showed that DLGAP1-AS2 knockdown and miR-154-5p overexpression inhibited cell invasion and migration, and the combination of LGAP1-AS2 knockdown and miR-154-5p overexpression showed stronger effects.

Conclusion: DLGAP1-AS2 knockdown may inhibit HCC cell migration and invasion by regulating miR-154-5p methylation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
A high level of DLGAP1-AS2 in HCC predicted poor survival. Expression of DLGAP1-AS2 in paired HCC and nontumor tissues was determined by RT-qPCR. Gene expression levels in paired tissues were expressed as average values of 3 technical replicates (a). ∗∗∗p < 0.001. With median level of DLGAP1-AS2 expression in HCC tissues as a cutoff value, the 62 patients were divided into high and low DLGAP1-AS2 level groups (n = 31). Survival curves were plotted and compared by the log-rank test (b).
Figure 2
Figure 2
miR-154-5p is downregulated in HCC and was inversely correlated with DLGAP1-AS2. Expression of miR-154-5p in paired HCC and nontumor tissues was determined by RT-qPCR. Gene expression levels in paired tissues were expressed as average values of 3 technical replicates (a). ∗∗∗p < 0.001. Linear regression was performed to analyze the correlations between miR-154-5p and DLGAP1-AS2 across HCC tissues (b), but not across nontumor tissues (c).
Figure 3
Figure 3
DLGAP1-AS2 knockdown upregulated miR-154-5p through methylation. To explore the interaction between DLGAP1-AS2 and miR-154-5p, SNU-398 cells were transfected with DLGAP1-AS2 siRNA or miR-154-5p mimic, followed by the confirmation of transfections by RT-qPCR (a). The effects of DLGAP1-AS2 siRNA silencing on miR-154-5p (b) or the effects of miR-154-5p overexpression on DLGAP1-AS2 (c) were also analyzed by RT-qPCR. MSP was performed to analyze the methylation of miR-154-5p in cells with DLGAP1-AS2 siRNA or NC siRNA transfection (d). Data of multiple transfection groups were expressed as mean ± SD values of 3 biological replicates. M: methylation; U: unmethylation. ∗p < 0.05.
Figure 4
Figure 4
DLGAP1-AS2 siRNA silencing and miR-154-5p overexpression inhibited cell invasion and migration. Transwell assays were performed to analyze the effects of DLGAP1-AS2 siRNA silencing and miR-154-5p overexpression on the cell invasion (a) and migration (b) of SNU-398 cells. Data of multiple transfection groups were expressed as mean ± SD values of 3 biological replicates. ∗p < 0.05.
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