Long non-coding RNA LUCAT1 promotes tumourigenesis by inhibiting ANXA2 phosphorylation in hepatocellular carcinoma

Abstract

Long non-coding RNAs (lncRNAs) play essential roles in diverse biological processes; however, current understanding of the mechanism underlying the regulation of tumour proliferation and metastasis is limited. Lung cancer-associated transcript 1 (LUCAT1) has been reported in a variety of human cancers, while its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to determine the biological role and underlying mechanism of LUCAT1 on progression and metastasis in HCC cells and clinical specimens. Our results demonstrated that LUCAT1 was up-regulated in HCC tissues and cells. Loss- and gain-of-function studies revealed that LUCAT1 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Furthermore, RNA pulldown and Western blot assays indicated that LUCAT1 inhibited the phosphorylation of Annexin A2 (ANXA2) to reduce the degradation of ANXA2-S100A10 heterotetramer (AIIt), which in turn accelerated the secretion of plasminogen into plasmin, thereby resulting in the activation of metalloprotease proteins. In conclusion, we propose that LUCAT1 serves as a novel diagnostic and therapeutic target for HCC.

Keywords: Annexin A2; hepatocellular carcinoma; long non-coding RNA; phosphorylation; tumour progression and metastasis.

Figure 1

Lung cancer associated transcript 1 (LUCAT1) is aberrantly up‐regulated in HCC tissues. (A) An increase in the expression of LUCAT1 occurs in hepatocellular carcinoma (HCC) tumour tissues compared to matched adjacent non‐tumour tissues (n = 90). Lung cancer associated transcript 1 expression was tested by quantitative real‐time PCR and measured using the $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_T}$ method, analysed with paired student's t test, and presented as means ± SEM. (B) Based on the median value of the LUCAT1 expression in HCC tissues, patients were divided into two groups (LUCAT1‐high expression group and LUCAT1‐low expression group), the Kaplan‐Meier survival analysis was used to calculate the overall survival. (C,D) The differential expression level of LUCAT1 in HCC cells, detected by quantitative reverse transcription‐PCR and northern blot assays. (E) RNA fluorescence in situ hybridization was conducted to detect the sub‐location of LUCAT1 (red) in MHCC97H cells, which revealed that it was mainly located in cytoplasm. A scale is presented at the lower right of the first panel. Magnification: 400×

Figure 2

Lung cancer associated transcript 1 (LUCAT1) enhances the proliferation of hepatocellular carcinoma (HCC) cells in vitro. (A) Proliferation rates were assessed using the CCK8 assay, and overexpression of LUCAT1 promoted HepG2 and Huh7 cell proliferation, whereas knockdown of LUCAT1 inhibited MHCC97H and Hep3B cell proliferation. (B) EdU immunofluorescence staining confirmed the function of LUCAT1 on HCC cell proliferation. Magnification: 100×. (C) The cell cycle distribution of HCC cells treated with LUCAT1 was assessed by flow cytometry. The distribution of cell cycle is shown in the graphs. All experiments were performed in triplicate and expressed as the mean ± SEM (*P < 0.05, ** P < 0.01)

Figure 3

Lung cancer associated transcript 1 (LUCAT1) enhances hepatocellular carcinoma (HCC) cell migration and invasion in vitro. (A) Wound healing assays were conducted to assess HCC cell migration. Representative wound healing images at 0, 24 and 48 h are shown. Quantitative analysis revealed that LUCAT1 significantly enhances HCC cell migration. Magnification: 40×. (B) Transwell assays of overexpressed and silenced LUCAT1 and their negative control HCC cells were performed, and migration and invasion were measured. The bar graph shows the number of cells that migrated or invaded the membrane. Magnification: 100×. All experiments were performed in triplicate and expressed as the mean ± SEM (*P < 0.05, ** P < 0.01)

Figure 4

Lung cancer associated transcript 1 (LUCAT1) enhances tumour growth and metastasis in vivo. (A‐F) BALB/c nude mice (6 wk of age) were subcutaneously transplanted with MHCC97H cells, sh‐LUCAT1‐MHCC97H cells into the left groin and sh‐NC‐MHCC97H cells into the right groin (four mice in each group). The volume of the tumours was calculated every 5 d after transplantation, and the mice were killed 40 d after implantation. Lung cancer associated transcript 1 strengthened MHCC97H cell growth in the tumours of the nude mice, which was confirmed by the staining of formalin‐fixed paraffin‐embedded of the subcutaneous tumour tissues with proliferating cell nuclear antigen (PCNA) Magnification: 200×. (F) In the tail vein xenograft model, mice (10 in each group) were injected with HepG2 cells (5 × 106 cells suspended in 200 μL phosphate buffered saline) through the tail vein, and lung metastasis was investigated respectively using the IVIS Lumina II system. (G) All the results of lung colonization were validated by the histological examination (H&E) Magnification: 100×. (H) Compared to the control group (three mice presented lung colonization), eight mice showed lung colonization with a higher number and larger tumours in the LUCAT1 overexpressing group. (*P < 0.05, ** P < 0.01)

Figure 5

Lung cancer associated transcript 1 (LUCAT1) interacts with Annexin A2 (ANXA2). (A) RNA pulldown assays were performed using biotin‐labelled sense or antisense LUCAT1 in MHCC97H cells. Sliver staining and mass spectrometry were conducted to identify the interacting proteins. The red arrow indicates the ANXA2 band. (B) Schematic map of the potential binding site of LUCAT1 on the ANXA2 protein. (C) RNA binding protein immunoprecipitation (RIP) assays were performed using an anti‐ANXA2 antibody and confirmed with agarose gel electrophoresis using a different probe. Fold increases were calculated by comparing with the input in the lower panel. (D) Antibody‐labelled ANXA2 (green) and probelabelled LUCAT1 (red) was detected in the MHCC97H cells. DAPI (blue) staining indicates nuclei. A scale is presented at the lower right of the first panel. Magnification: 400×. Data are expressed as the means ± SEM

Figure 6

Lung cancer associated transcript 1 (LUCAT1) inhibits the phosphorylation of Annexin A2 (ANXA2) to accelerate plasminogen into plasmin. (A) Co‐immunoprecipitation (Co‐IP) assays were performed with S100A10 antibody in ANXA2 overexpressing cells. (B) Annexin A2 and pSer25ANXA2 expression levels were investigated in LUCAT1 overexpressed and deleted cells. (C,D) Co‐immunoprecipitation assays showed that ANXA2 precipitated less S100A10 in LUCAT1 knockdown cells (C) and more S100A10 in LUCAT1 overexpressing cells (D). (E,F) ELISA assays revealed the influence of LUCAT1 on the expression of plasmin and MMP‐9. (G) epithelial–mesenchymal transition‐ and cell cycle‐related proteins were investigated in LUCAT1 overexpressed and deleted cells

Figure 7

Schematic diagram illustrating that Lung cancer associated transcript 1 (LUCAT1) inhibits the phosphorylation of Annexin A2 (ANXA2) at Ser‐25, further suppressing the degradation of AIIt, and then accelerating the conversion of plasminogen into plasmin, which activates metalloprotease proteins, leading to the progression and metastasis of HCC