Long non-coding RNA DUXAP8 promotes tumorigenesis by regulating IGF1R via miR-9-3p in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide with a low 5-year survival rate. Long non-coding RNA (lncRNA) double homeobox A pseudogene 8 (DUXAP8) is an oncogene and a potential biomarker in various tumors, such as ovarian, colorectal and non-small-cell lung cancer. However, the function and molecular mechanism underlying DUXAP8 in HCC progression is not completely understood. The expression of DUXAP8, microRNA (miR)-9-3p and insulin-like growth factor 1 receptor (IGF1R) in HCC tissues and cells was detected via reverse transcription-quantitative PCR. The expression levels of IGF1R and epithelial-mesenchymal transition-associated proteins (Snail, Slug, E-cadherin, N-cadherin and vimentin) were assessed via western blotting. The effects of DUXAP8, miR-9-3p and IGF1R on proliferation, migration and invasion were examined by conducting Cell Counting Kit-8 and Transwell assays, respectively. The interaction between miR-9-3p and DUXAP8 or IGF1R was predicted using StarBase or TargetScan, and further assessed using dual luciferase reporter and RNA immunoprecipitation assays. DUXAP8 and IGF1R were upregulated and miR-9-3p was downregulated in HCC tissues and cells compared with adjacent healthy tissues and a normal liver cell line, respectively. miR-9-3p overexpression decreased the protein expression level of IGF1R, and miR-9-3p knockdown enhanced the protein expression level of IGF1R in HCC cells compared with the corresponding control groups. Moreover, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression increased E-cadherin protein expression levels, and decreased Snail, Slug, N-cadherin and vimentin protein expression levels. However, miR-9-3p inhibitor and IGF1R overexpression reversed DUXAP8 knockdown- and miR-9-3p overexpression-induced effects, respectively. In addition, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression suppressed proliferation, migration and invasion, which was reversed by miR-9-3p inhibitor and IGF1R overexpression, respectively. Moreover, miR-9-3p as the target of DUXAP8 and IGF1R as the target of miR-9-3p were verified in HCC cells. lncRNA DUXAP8 contributed to HCC tumorigenesis via the miR-9-3p/IGF1R axis, providing a novel therapeutic approach for HCC diagnosis and treatment.

Keywords: epithelial-mesenchymal transition; hepatocellular carcinoma; insulin-like growth factor 1 receptor; invasion; long non-coding RNA double homeobox A pseudogene 8; microRNA-9-3p; migration; proliferation.

Figure 1

Expression patterns of DUXAP8 in HCC tissues and cells. (A) DUXAP8 expression levels in 38 paired HCC tumor and adjacent healthy tissues. (B) DUXAP8 expression levels in HCC cell lines (SK-HEP-1, Huh-7, Hep38 and Huh-1) and a normal human liver cell line (THLE-2). (C) Kaplan-Meier overall survival curves of patients with HCC based on the median expression of DUXAP8 expression levels. n=3. ^**P<0.01. DUXAP8, double homeobox A pseudogene 8; HCC, hepatocellular carcinoma.

Figure 2

DUXAP8 induces HCC cell proliferation, migration, invasion and EMT. (A) Knockdown efficiency of si-DUXAP8 in SK-HEP-1 and Huh-7 cells. Cell proliferation in si-DUXAP8-transfected (B) SK-HEP-1 and (C) Huh-7 cells. Cell (D) migration and (E) invasion in si-DUXAP8-transfected SK-HEP-1 and Huh-7 cells. (F) Expression levels of EMT-related proteins (Snail, Slug, E-cadherin, N-cadherin and vimentin) were detected via western blotting in si-DUXAP8-transfected SK-HEP-1 and Huh-7 cells. n=3. ^**P<0.01. DUXAP8, double homeobox A pseudogene 8; HCC, hepatocellular carcinoma; EMT, epithelial-mesenchymal transition; si, small interfering RNA; NC, negative control; OD, optical density.

Figure 3

miR-9-3p is a direct target of DUXAP8 in HCC cells. (A) Expression of miR-9-3p in 38 paired HCC tumor and adjacent healthy tissues. (B) Expression of miR-9-3p in HCC cell lines (SK-HEP-1, Huh-7, Hep38 and Huh-1) and a normal human liver cell line (THLE-2). (C) Correlation between DUXAP8 and miR-9-3p in HCC tissues. (D) Binding sites between DUXAP8 and miR-9-3p. The relative luciferase activity in (E) SK-HEP-1 and (F) Huh-7 cells co-transfected with DUXAP8-WT or DUXAP8-MUT vectors and miR-NC or miR-9-3p mimics. An RNA immunoprecipitation assay was conducted in (G) SK-HEP-1 and (H) Huh-7 cell extracts to examine whether miR-9-3p endogenously associated with DUXAP8. The effects of DUXAP8 (I) knockdown and (J) overexpression on miR-9-3p expression were detected via reverse transcription-quantitative PCR in SK-HEP-1 and Huh-7 cells. n=3. ^**P<0.01. miR, microRNA; DUXAP8, double homeobox A pseudogene 8; HCC, hepatocellular carcinoma; WT, wild-type; MUT, mutant; NC, negative control; si, small interfering RNA; Ago2, Argonaute2.

Figure 4

DUXAP8 facilitates HCC progression by targeting miR-9-3p. (A) miR-9-3p expression levels in SK-HEP-1 and Huh-7 cells transfected with si-NC, si-DUXAP8, si-DUXAP8 + anti-miR-NC or si-DUXAP8 + miR-9-3p inhibitor. Cell proliferation in (B) SK-HEP-1 and (C) Huh-7 cells. Cell (D) migration and (E) invasion in transfected SK-HEP-1 and Huh-7 cells. EMT-related protein expression levels were (F) determined by western blotting and (G) semi-quantified in SK-HEP-1 cells. EMT-related protein expression levels were (H) determined by western blotting and semi-quantified in Huh-7 cells. n=3. ^*P<0.05; ^**P<0.01. DUXAP8, double homeobox A pseudogene 8; HCC, hepatocellular carcinoma; miR, microRNA; si, small interfering RNA; NC, negative control.

Figure 5

IGF1R is a target of miR-9-3p. (A) Putative binding sequences between miR-9-3p and IGF1R. The relative luciferase activity of (B) SK-HEP-1 and (C) Huh-7 cells co-transfected with IGF1R 3'UTR-WT or IGF1R 3'UTR-MUT vectors and miR-NC or miR-9-3p mimics. The effects of miR-9-3p (D) overexpression and (E) knockdown on IGF1R expression in SK-HEP-1 and Huh-7 cells. IGF1R expression levels in SK-HEP-1 and Huh-7 cells transfected with (F) miR-NC, miR-9-3p mimics, (G) anti-miR-NC or miR-9-3p inhibitor. (H) Expression of IGF1R in 38 paired HCC tumor and adjacent healthy tissues. (I) Correlation between IGF1R and miR-9-3p expression in HCC tumor tissues. n=3. ^**P<0.01. IGF1R, insulin-like growth factor 1 receptor; miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant; NC, negative control; HCC, hepatocellular carcinoma.

Figure 6

miR-9-3p suppresses HCC progression by targeting IGF1R. (A) IGF1R expression in SK-HEP-1 and Huh-7 cells. IGF1R protein expression levels in (B) SK-HEP-1 and (C) Huh-7 cells. Cell proliferation in (D) SK-HEP-1 and (E) Huh-7 cells transfected with miR-NC, miR-9-3p mimics, miR-9-3p mimics + pcDNA3.1 or miR-9-3p mimics + IGF1R. Cell (F) migration and (G) invasion in SK-HEP-1 and Huh-7 cells transfected with miR-NC, miR-9-3p mimics, miR-9-3p mimics + pcDNA3.1 or miR-9-3p mimics + IGF1R. (H and I) EMT-related protein expression levels were (H) determined by western blotting and (I) semi-quantified in SK-HEP-1 cells. EMT-related protein expression levels were (J) determined by western blotting and (K) semi-quantified in Huh-7 cells. n=3. ^*P<0.05; ^**P<0.01. miR, microRNA; HCC, hepatocellular carcinoma; IGF1R, insulin-like growth factor 1 receptor; NC, negative control; EMT, epithelial-mesenchymal transition; OD, optical density.

Figure 7

DUXAP8 regulates IGF1R expression by sponging miR-9-3p. (A) Correlation between DUXAP8 and IGF1R expression in hepatocellular carcinoma tumor tissues. (B) IGF1R expression in SK-HEP-1 and Huh-7 cells transfected with si-NC, si-DUXAP8, si-DUXAP8 + anti-miR-NC and si-DUXAP8 + miR-9-3p inhibitor. IGF1R protein expression levels in transfected (C) SK-HEP-1 and (D) Huh-7 cells. n=3. ^**P<0.01. DUXAP8, double homeobox A pseudogene 8; IGF1R, insulin-like growth factor 1 receptor; miR, microRNA; si, small interfering RNA; NC, negative control.