STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4

Abstract

Background: Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in hepatocellular carcinoma (HCC) remains poorly understood.

Methods: LncRNA-HOXD-AS1 was identified by microarray and validated by real-time PCR. The clinicopathological significance of HOXD-AS1 was analyzed by Kaplan-Meier method. Chromatin immunoprecipitation was conducted to examine the mechanism of HOXD-AS1 upregulation. The role of HOXD-AS1 in HCC cells was assessed both in vitro and in vivo. ceRNA function of HOXD-AS1 was evaluated by RNA immunoprecipitation and biotin-coupled miRNA pull down assays.

Results: In this study, we found that HOXD-AS1 was significantly upregulated in HCC tissues. Clinical investigation demonstrated high expression level of HOXD-AS1 was associated with poor prognosis and high tumor node metastasis stage of HCC patients, and was an independent risk factor for survival. Moreover, our results revealed that STAT3 could specifically interact with the promoter of HOXD-AS1 and activate HOXD-AS1 transcription. Knockdown of HOXD-AS1 significantly inhibited migration and invasion of HCC cells in vitro and distant lung metastasis in vivo. Additionally, HOXD-AS1 was enriched in the cytoplasm, and shared miRNA response elements with SOX4. Overexpression of HOXD-AS1 competitively bound to miR-130a-3p that prevented SOX4 from miRNA-mediated degradation, thus activated the expression of EZH2 and MMP2 and facilitated HCC metastasis.

Conclusions: In summary, HOXD-AS1 is a prognostic marker for HCC patients and it may play a pro-metastatic role in hepatocarcinogenesis.

Keywords: HOXD-AS1; Hepatocellular carcinoma; Metastasis; SOX4; ceRNA.

Fig. 1

HOXD-AS1 is upregulated and positively associated with poor prognosis in HCC patients. a Hierarchical clustering analysis of 26 lncRNAs that were differentially expressed in 14 paired samples of cancerous tissues (c) and corresponding adjacent noncancerous liver tissues (N) (fold change > 2² or <2^-2, p < 0.05). Expression values are represented in shades of red and green. b The fold change of HOXD-AS1 expression in 14 HCC tissues (probe: 228601_at). c Left: HOXD-AS1 expression in 89 paired samples of HCC tissues (p < 0.0001). Right: Pie chart of the proportions of HCC samples in which HOXD-AS1 expression was upregulated (blue), downregulated (red), or showed no change (green). d Correlation between HOXD-AS1 expression and overall survival in 120 HCC patients. Median value of HOXD-AS1 expression was chosen as the point for separating tumors with low or high-level expression group. (Log-rank; p = 0.0179). e The expression levels of HOXD-AS1 in nuclear and cytoplasm of HCC cells. U1 (nuclear retained) and GAPDH (exported to cytoplasm) were used as controls. f Fluorescence in situ hybridization analysis of the subcellular location of HOXD-AS1 in HCC cells

Fig. 2

HOXD-AS1 is regulated by the transcription factor STAT3. a mRNA expression level of STAT3 in 68 paired samples of HCC tissues. b Expression correlation analysis between HOXD-AS1 (x) and STAT3 (y) in 68 HCC tissues (R = 0.4743, p < 0.0001). c Expression levels of HOXD-AS1 in Hep3B and Huh7 cells after knockdown of STAT3. d Left: schematic representation of constructs for HOXD-AS1 promoter reporter. Right: luciferase activities of 6 truncated constructs in HEK-293 T cells. e Luciferase activities of -1158 bp ~ -746 bp or -2000 bp ~ -1159 bp fragments after knockdown of STAT3. f ChIP assay in HCC cells, followed by quantitative PCR amplification of binding site 2 within HOXD-AS1 promoter region. Genomic DNA input was 1%. Data in (a-e) are the means ± SD. **p < 0.01, and ***p < 0.001

Fig. 3

HOXD-AS1 promotes invasion and metastasis of HCC cells in vitro and in vivo. a, b Migration and invasion analyses of SMCC-7721 and Huh7 cells with shRNA knockdown of HOXD-AS1. Representative images are shown on the left, and the average number of cells per field at the indicated time points is shown on the right. c, d Migration and invasion assays of Huh28 and SNU449 cells were performed after transduction with HOXD-AS1 or control lentivirus, respectively. e Gross morphology of representative lungs and characteristic H&E staining of metastatic nodules in the lung of nude mice 6 weeks after tail vein injection with tumor cells. The numbers of metastatic nodules in the lungs of each mouse (n = 12 mice per group) were counted and analyzed using. Data in A-E are the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

HOXD-AS1 is involved in migration of HCC cells through regulating SOX4. a Expression correlation between HOXD-AS1 (x) and SOX4 (y) in 14 HCC tissues. Data are depicted as probe signal value of global gene expression microarray (R = 0.5807, p = 0.0012). b, c The expression of SOX4 in Huh7 cells with HOXD-AS1 knockdown and SNU449 cells with HOXD-AS1 overexpression. β-actin was used as the loading control. d Migration assays of Huh7 cells with HOXD-AS1 knockdown and SOX4 overexpression. e Migration assays of SNU449 cells with HOXD-AS1 overexpression and SOX4 knockdown. Data in b-e are the means ± SD. *p < 0.05 and **p < 0.01

Fig. 5

HOXD-AS1 regulates SOX4 expression through a miRNA-dependent mechanism. a. RIP experiments were performed using the AGO2 antibody, and specific primers were used to detect the enrichment of HOXD-AS1 and SOX4. The data represent the average and standard deviation of three independent experiments. b Dicer deficiency rescued the siHOXD-AS1-mediated reduction of HOXD-AS1 and SOX4 mRNA in Huh7 cells. c Firefly luciferase activity normalized to Renilla luciferase activity in HEK-293 T cells co-transfected with luciferase reporters with wild type or mutant transcripts of HOXD-AS1 or SOX4 along with miR-130a-3p mimics or negative control (NC). d Protein levels of SOX4 following transfection of miR-130a-3p mimics or NC into Huh-7 and SMMC-7721 cells. e mRNA level of HOXD-AS1 was detected by biotin-coupled miR-130a-3p pull-down in Huh7 and SMMC-7721 cells. f Left: Protein level of SOX4 after transfection of miR-130a-3p mimics in SNU449 cells with HOXD-AS1 overexpression. Right: Protein level of SOX4 after transfection of miR-130a-3p inhibitors in Huh7 cells with HOXD-AS1 knockdown. Data in (b, c, e) are the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 6

SOX4 is required for MMP2 and EZH2 expression mediated by HOXD-AS1. a mRNA and protein levels of metastasis-related genes in Huh7 cells with HOXD-AS1 knockdown. b mRNA and protein levels of metastasis-related genes in SNU449 cells with HOXD-AS1 overexpression. c The expression level change of EZH2 and MMP2 after knockdown of SOX4 in SNU449 cells with HOXD-AS1 overexpression. d ChIP assay was performed with antibody against SOX4 or control IgG in Huh28 cells with HOXD-AS1 overexpression or negative control. Immunoprecipitated DNA was analyzed by quantitative PCR. Genomic DNA input was 1%. e A proposed model for illustrating the function and mechanism of HOXD-AS1 in HCC metastasis. Data in A-D are the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001