Long noncoding RNA DLX6-AS1 promotes liver cancer by increasing the expression of WEE1 via targeting miR-424-5p

Abstract

Long noncoding RNAs (lncRNAs) played an important role in tumorigenesis and development of hepatocellular carcinoma (HCC). In this study, we first demonstrated that lncRNA DLX6 antisense RNA 1 (DLX6-AS1) was upregulated in cancer tissues and cells lines compared with normal adjacent and cell line. Knock-down DLX6-AS1 by transfection with small interfering RNA (siRNA) suppressed cell proliferation, migration, and invasion of HCC cells. Cell cycle analysis showed that cells transfected with siRNA were arrested in G0/G1 phase. Then, we performed dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay to show that DLX6-AS1 could bind with miR-424-5p. And cotransfection inhibitor of miR-424-5p with siRNA of DLX6-AS1 could abolish the inhibitory effect of siRNA of DLX6-AS1 on cell proliferation, migration, and invasion. Moreover, we further demonstrated that the oncogene WEE1 G2 checkpoint kinase (WEE1) was the target of miR-424-5p and expression levels of WEE1 were positive correlation with that of DLX6-AS1. Taken together, these results suggested that upregulated DLX6-AS1 promoted cell proliferation, migration, and invasion of HCC through increasing expression of WEE1 via targeting miR-424-5p.

Keywords: DLX6-AS1; WEE1; hepatocellular carcinoma; long noncoding RNA; miR-424-5p.

Figure 1

The expression of DLX6‐AS1 in the HCC tissues and cell lines as determined by qRT‐PCR. A, The expression of DLX6‐AS1 is upregulated in the HCC cancer tissues compared with the adjacent normal tissues. B, Upregulated DLX6‐AS1 in the HCC cell lines compared with normal human liver cell line (LO2). *P < 0.05; ***P < 0.001. HCC, hepatocellular carcinoma; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction

Figure 2

Cellular function of DLX6‐AS1 in SK‐HEP‐1 and Hep3B. A, DLX6‐AS1 is knocked‐down in cells transfected with siRNAs. B, Growth curves were obtained by MTT assay. Decreased growth rate in cells transfected with siDLX6‐AS1#3. C, Representative images of colony formation assay and the number of colonies were counted. D, Cell cycles were analyzed by flow cytometry. Transfection with siDLX6‐AS1#3 changes the cell cycle distribution. E, Representative images of wound healing assay and cell mobility are shown. Transfection with siDLX6‐AS1 decreases cell migration. F, Representative images of the matrigel transwell assay and cells invaded through matrigel were counted. ***P < 0.001. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; siRNA, small interfering RNA

Figure 3

DLX6‐AS1 targets miR‐424‐5p. A, The predicted miR‐424‐5p binding sites on the 3′‐UTR of DLX6‐AS1 are shown. B, Dual‐luciferase reporter assay were performed and mutations in the DLX6‐AS1 binding site abolish the inhibitory effect of miR‐424‐5p mimics on the luciferase activity. C, The expression levels of miR‐424‐5p and DLX6‐AS1 were determined by qRT‐PCR in Ago2‐immunoprecipitated complex, and miR‐424‐5p and DLX6‐AS1 are enriched compared with positive control (input) and negative control (anti‐IgG). The protein levels of Ago were determined to show efficient immunoprecipitation of anti‐Ago from cell extracts. D, The expression levels of DLX6‐AS1 were determined by qRT‐PCR in the complex which were pulled down by biotinylated miR‐424‐5p mimics, mutant miR‐424‐5p mimics or NC. ***P < 0.001. 3′‐UTR, 3′‐untranslated region; Mut, mutant; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; Wt, wild‐type

Figure 4

Transfection with miR‐424‐5p inhibitor abolishes the effect of siDLX6‐AS1. A, Growth curves were obtained by MTT assay. B, Representative images of colony formation assay are shown and the numbers of colonies were counted. C, Cell cycle distribution was determined by flow cytometry. D, Representative images of wound healing assay are shown and cell mobility was calculated. Transfection with siDLX6‐AS1 decreases cell migration. E, Representative images of matrigel transwell assay and cells invaded through matrigel were counted. ***P < 0.001. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide

Figure 5

miR‐424‐5p targets the 3′‐UTR of WEE1. A, The predicted miR‐424‐5p binding sites in the 3′‐UTR of WEE1 are shown. B, Dual‐luciferase reporter assay was performed and mutations in the 3′‐UTR of WEE1 abolish the inhibitory effect of miR‐424‐5p mimics on the luciferase activity. C, D, The expression levels of WEE1 are decreased/increased in SK‐HEP‐1 and Hep3B cells transfected with mimics/inhibitor of miR‐424‐5p as determined by Western blot analysis and qRT‐PCR. E, F, The expression levels of WEE1 are increased/decreased in SK‐HEP‐1 and Hep3B cells transfected with DLX6‐AS1 overexpression plasmid/siRNA as determined by Western blot analysis and qRT‐PCR. G, Correlations among the expression of DLX6‐AS1, miR‐424‐5p, and WEE1 were analyzed with a Spearman's rank correlation. ***P < 0.001. 3′‐UTR, 3′‐untranslated region; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; siRNA, small interfering RNA