Knockdown of LncRNA ANRIL suppresses cell proliferation, metastasis, and invasion via regulating miR-122-5p expression in hepatocellular carcinoma

Abstract

Objective: Previous studies reported that lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) was upregulated in hepatocellular carcinoma (HCC) tissues and decreased expression of ANRIL could suppress cell proliferation, metastasis, and invasion and induce apoptosis of HCC cells. However, the molecular mechanism of ANRIL involved in HCC tumorigenesis is still unknown.

Methods: The expressions of ANRIL and miR-122-5p in HCC tissues and cells were quantified by qRT-PCR. MTT assay, colony formation assay, wound healing assay, and transwell invasion assay were performed to evaluate cell growth, metastasis, and invasion, respectively. RNA immunoprecipitation (RIP) assay and luciferase reporter assay were performed to determine whether ANRIL could directly bind to miR-122-5p in HCC cells. Xenograft tumor experiment was conducted to confirm the biological role and underlying mechanism of ANRIL in vivo.

Results: The results showed that ANRIL was upregulated and miR-122-5p was downregulated in HCC tissues and cells. ANRIL was negatively correlated with miR-122-5p expression in HCC tissues. Knockdown of ANRIL or miR-122-5p overexpression suppressed HCC cell viability, colony formation ability, metastasis, and invasion. ANRIL was demonstrated to directly bind to miR-122-5p and inhibit its expression. Forced expression of ANRIL abolished the inhibitory effect of miR-122-5p overexpression on HCC progression. In vivo experiment demonstrated that ANRIL knockdown impeded tumor growth in vivo and increased miR-122-5p expression.

Conclusion: Our finding suggested that knockdown of ANRIL suppressed cell proliferation, metastasis and invasion via regulating miR-122-5p expression in HCC, illustrating the underlying mechanism of the oncogenic role of ANRIL in HCC.

Keywords: ANRIL; HCC; Invasion; Metastasis; MiR-122-5p; Proliferation.

Fig. 1

Expression levels of ANRIL and miR-122-5p in HCC tissues. qRT-PCR analysis was conducted to assess the expression levels of ANRIL (a) and miR-122-5p (b) in 31 paired HCC tissues and their corresponding adjacent normal tissues. c Negative correlation between ANRIL and miR-122-5p expression

Fig. 2

Expression levels of ANRIL and miR-122-5p in HCC cell lines. qRT-PCR was employed to examine the expression levels of ANRIL and miR-122-5p in HCC cell lines (SMMC7721, HUH7, Hep3B, and HepG2) and normal hepatic cell line LO2. *P < 0.05

Fig. 3

ANRIL knockdown significantly suppressed HCC cell growth, metastasis, and invasion. HepG2 and SMMC7721 cells were transfected with si-ANRIL or si-NC and cultured for 48 h. a MTT assay was used to detect cell viability of transfected HepG2 and SMMC7721 cells. b Colony formation assay was performed to assess clonogenic potential of transfected HepG2 and SMMC7721 cells. c Wound healing assay was conducted to evaluate the metastatic capacity of transfected HepG2 and SMMC7721 cells. d Transwell invasion assay was implemented to determine cell invasive ability of transfected HepG2 and SMMC7721 cells. *P < 0.05

Fig. 4

ANRIL functioned as a molecular sponge of miR-122-5p in HCC. a Predicted wild type or mutated miR-122-5p-binding sites in ANRIL. b MS2-RIP followed by qRT-PCR to detect miRNAs endogenously associated with ANRIL in HepG2 and SMMC7721 cells. c Luciferase activity was determined by luciferase reporter assay in HepG2 and SMMC7721 cells cotransfected with pmirGLO, pmirGLO-ANRIL-WT, or pmirGLO-ANRIL-MUT and miR-122-5p or miR-NC. d Expression level of miR-122-5p in HepG2 and SMMC7721 cells transfected with si-ANRIL, pcDNA-ANRIL, or respective controls was evaluated by qRT-PCR. *P < 0.05

Fig. 5

ANRIL overexpression reversed the inhibitory effect of miR-122-5p on HCC cell proliferation, metastasis, and invasion. HepG2 cells were transfected with miR-NC, miR-122-5p, or combined with pcDNA or pcDNA-ANRIL and cultured for 48 h. a Cell viability in transfected HepG2 cells was determined by MTT assay. b Colony number of transfected HepG2 cells was detected by colony formation assay. c Wound healing assay was conducted to assess cell metastasis in transfected HepG2 cells. d Transwell invasion assay was applied to examine cell invasive ability of transfected HepG2 cells. *P < 0.05

Fig. 6

ANRIL knockdown suppressed tumor growth and induced miR-122-5p expression in HCC cells in vivo. HepG2 cells (1 × 10⁶) transfected with sh-ANRIL or sh-NC were subcutaneously injected into either side of flank area of nude mice (n = 4 per group) and then housed in specific pathogen free facilities. a Tumor volumes were measured once every 5 days for 35 days. b Mice were killed at 35 days after injection and xenografts were dissected and weighted. c Relative expression of miR-122-5p in tumor xenograft tissues was examined by qRT-PCR. *P < 0.05