Pseudogene DUXAP8 Promotes Cell Proliferation and Migration of Hepatocellular Carcinoma by Sponging MiR-490-5p to Induce BUB1 Expression

Abstract

Hepatocellular Carcinoma (HCC) currently remains one of the most lethal malignancies worldwide. Recently, long non-coding RNAs (lncRNAs) had been demonstrated to play a crucial role in the progression of multiple human cancers, including HCC. In this study, we found that lncRNA DUXAP8 was upregulated in tumor samples and served as an oncogene in HCC. Bioinformatics analysis showed that DUXAP8 was significantly associated with the regulation of centrosome organization, homologous recombination, meiotic cell cycle process, sister chromatid segregation, nuclear chromosome segregation, and RNA export from the nucleus. The knockdown of DUXAP8 significantly suppresses cell proliferation and the cell cycle but induces cell apoptosis in HCC. Mechanically, the present study showed that DUXAP8 serves as a sponge of MiR-490-5p to promote the expression of BUB1 in HCC. Although the underlying regulatory mechanisms of DUXAP8 in HCC require further investigation, this study, for the first time, showed that DUXAP8 can serve as a new therapeutic target for HCC.

Keywords: Hepatocellular Carcinoma; bioinformatics; long non-coding RNA; regulatory mechanism; target.

FIGURE 1

DUXAP8 was upregulated in HCC. (A,B) DUXAP8 expression in HCC tissues was found to be higher than in normal tissue by analyzing GSE84402 and GSE121248. (C) DUXAP8 expression in HCC tissues was found to be higher than in normal tissue by analyzing TCGA. (D) DUXAP8 expression levels were upregulated in Stage II/III compared to Stage I samples. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

Upregulation of DUXAP8 expression is associated with poor prognosis of HCC. (A) Upregulation of DUXAP8 expression is associated with shorter OS of HCC. (B–I) increased DUXAP8 expression predicted poor OS in patients with low mutation burden (B), high mutation burden (C), Grade I (D), Grade II (E), Grade III (F), Stage I (G), Stage II (H), and Stage III (I) HCC.

FIGURE 3

Bioinformatics analysis of DUXAP8 in HCC.

FIGURE 4

(A) Construction of the DUXAP8-mediated PPI network in HCC. (B) GO and KEGG pathway analysis of the DUXAP8-mediated hub PPI network in HCC.

FIGURE 5

Knockdown of DUXAP8 suppressed HCC Cell Viability. (A,C) The endogenous DUXAP8 expressions in Huh-7 (A) and HepG2 (C) cells transfected with siDUXAP8 were reduced. (B,D) The results showed that the silencing of DUXAP8 significantly suppressed cell proliferation in both Huh-7 (B) and HepG2 (D). (E,F) The results showed that the silencing of DUXAP8 significantly suppressed the cell cycle in Huh-7 cells. (G,H) The results showed that the silencing of DUXAP8 significantly suppressed the cell cycle in HepG2 cells. All assays were conducted with at least three determinations. Statistical comparisons between groups of normalized data were performed using Students’ t-test. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 6

Knockdown of DUXAP8 induced HCC cell apoptosis. (A,B) The results showed that the silencing of DUXAP8 significantly induced cell apoptosis in Huh-7 cells. (C,D) The results showed that the silencing of DUXAP8 significantly induced cell apoptosis in HepG2 cells. All assays were conducted with at least three determinations. Statistical comparisons between groups of normalized data were performed using Students’ t-test. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 7

Knockdown of DUXAP8 suppressed Cell cycle regulators. (A,B) Knockdown of DUXAP8 suppressed the expression of Cell cycle regulators in both Huh-7 (A) and HepG2 (B). All assays were conducted at least three determinations. Statistical comparisons between groups of normalized data were performed using Students’ t-test. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 8

DUXAP8 promoted BUB1 expression through sponging MiR-490-5p. (A,B) The RNA levels of BUB1 and DUXAP8 were significantly reduced after overexpressing miR-490-5p in Huh-7 (A) and HepG2 (B) cells by using RT-PCR assay. (C) Western blot assay also showed that the protein levels of BUB1 were significantly decreased in Huh-7 cells transfected with miR-490-5p compared to the control group. (D) Bioinformatics analysis predicted the direct interaction between miR-490-5p and BUB1 or DUXAP8. (E,F) Luciferase reporter assay revealed that luciferase activity was significantly repressed in the constructs of the DUXAP8 (E)- and BUB1-3′UTR (F) when co-transfected with the corresponding miRNAs compared with NC, whereas the mutated 3′UTR did not show a significant response to miR-490-5p. (G) The knockown efficiency of MiR-490-5p in HCC cells. (H) The expression levels of BUB1 after knockdown of DUXAP8 or miR-490-5p. All assays were conducted at with least three determinations. Statistical comparisons between groups of normalized data were performed using Students’ t-test. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 9

Overexpresion of BUB1 reverses the effects of DUXAP8 knockdown in HCC. (A,B) Knockdown of BUB1 decreased cell proliferation in Huh-7 and HepG2. (C,D) Overexpression of BUB1 reversed the effects of decreased cell proliferation in DUXAP8 knockdown Huh-7 and HepG2 cells. All assays were conducted with at least three determinations. Statistical comparisons between groups of normalized data were performed using Students’ t-test. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).