Long non-coding RNA LINC00467 regulates hepatocellular carcinoma progression by modulating miR-9-5p/PPARA expression

Abstract

The aim of this study was to analyse the expression pattern and elucidate the mechanistic involvement of long non-coding RNA LINC00467 in hepatocellular carcinoma (HCC). The relative expression of LINC00467 and microRNA (miR)-9-5p was determined by real-time polymerase chain reaction. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell proliferation was analysed by cell counting. Cell migration and invasion were monitored by Transwell assay. The luciferase reporter assay was employed to investigate the regulatory effect of miR-9-5p on LINC00467 and peroxisome proliferator-activated receptor alpha (PPARA). The endogenous PPARA protein was quantified by western blotting. It was found that LINC00467 was aberrantly decreased in HCC. The ectopic expression of LINC00467 significantly suppressed cell viability, proliferation, migration and invasion. LINC00467 functioned as a sponge for miR-9-5a and negatively regulated miR-9-5p expression. We also identified PPARA as the direct target of miR-9-5p. siRNA-mediated knockdown of PPARA in LINC00467-proficient cells promoted cell viability, migration and invasion. Our data indicate the critical involvement of LINC00467/miR-9-5p/PPARA signalling in the incidence and progression of HCC.

Keywords: LINC00467; PPARA; hepatocellular carcinoma; miR-9-5p; microRNA sponge.

Figure 1.

The expression of LINC00467 in HCC clinical samples was analysed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). (a) LINC00467 levels were measured via qRT-PCR and normalized to the level of GAPDH in HCC tissues and adjacent non-cancerous tissues. (b) LINC00467 levels were measured via qRT-PCR and normalized to the level of GAPDH in HCC tissues with or without lymphatic metastasis. (**p < 0.01.)

Figure 2.

LINC00467 suppresses cell viability and proliferation in HCC cells. (a) LINC00467 levels were measured via qRT-PCR and normalized to the level of GAPDH in SMMC-7721 and HepG2 cells. (b) SMMC-7721 and HepG2 cells subjected to MTT assay after plating for 48 h. (c) SMMC-7721 and (d) HepG2 cells were subjected to cell number assay every 24 h. (**p < 0.01.)

Figure 3.

LINC00467 suppresses cell migration and invasion in HCC cells. SMMC-7721 cells were subjected to Transwell migration assay (a) and invasion assay (b). HepG2 cells were subjected to Transwell migration assay (c) and invasion assay (d). Data are the mean ± s.d. of three independent experiments and each is measured in triplicate (**p < 0.01).

Figure 4.

LINC00467 is a molecular sponge for miR-9-5p. (a) Schematic showing the putative miR-9-5p target site in LINC00467. (b,c) Dual-luciferase assays showing the repression of LINC00467 following the transfection of synthetic miRNA precursor. Renilla luciferase activity was normalized to firefly activity and is presented as relative luciferase activity. (d) miRNA levels were measured via qRT-PCR and normalized to the level of U6. (e) LINC00467 levels were measured via qRT-PCR and normalized to the level of GAPDH following the transfection of the synthetic miRNA precursor. Data are the mean ± s.d. of three independent experiments and each is measured in triplicate (*p < 0.05, **p < 0.01).

Figure 5.

LINC00467 relieves the inhibitory effect of miRNA on PPARA. (a) The potential miR-9-5p binding site at the 3′UTR of PPARA. The target site mutations are underlined. (b,c) Dual-luciferase assays showing the repression of 3′UTR following the transfection of synthetic miRNA precursor. (d) Western blot analysis of PPARA expression following the transfection of synthetic miRNA precursor or NC. β-actin serves as an internal control. (e) Western blot analysis of PPARA expression in endometrial carcinoma cells which stably transfected pcDNA3.1-vec or pcDNA3.1-LINC00467. β-actin serves as an internal control. Data are the mean ± s.d. of three independent experiments and each is measured in triplicate (**p < 0.01).

Figure 6.

PPARA is critical for the function of LINC00467 in HCC. (a) SMMC-7721 and HepG2 cells subjected to MTT assay after plating for 48 h. (b) SMMC-7721 and (c) HepG2 cells were subjected to cell number assay every 24 h. (d) SMMC-7721 and HepG2 cells were subjected to Transwell migration assay. (e) SMMC-7721 and HepG2 cells were subjected to Transwell invasion assay. Data are the mean ± s.d. of three independent experiments and each is measured in triplicate (*p < 0.05, **p < 0.01).