LncRNA NEAT1 Promotes Proliferation, Migration And Invasion Via Regulating miR-296-5p/CNN2 Axis In Hepatocellular Carcinoma Cells

Abstract

Background: Emerging evidence has revealed that long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is implicated in the development of various cancers. However, the underlying molecular mechanisms of NEAT1 in hepatocellular carcinoma (HCC) remain unclear.

Methods: The expression of NEAT1, miR-296-5p and Calponin 2 (CNN2) was detected by quantitative real-time polymerase chain reaction or Western blot, respectively. Cell proliferation and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or flow cytometry, respectively. Transwell assay was used to determine cell migration and invasion. The interaction between miR-296-5p and NEAT1 or CNN2 was analyzed by dual-luciferase reporter assay and RIP assay. Huh7 cells transfected with sh-NEAT1 were used to establish the murine xenograft model.

Results: NEAT1 was elevated in HCC tissues and cell lines. Knockdown of NEAT1 significantly inhibited proliferation, migration and invasion of HCC cells in vitro as well as tumor growth in vivo. NEAT1 was a sponge of miR-296-5p and remarkably reduced the level of miR-296-5p in HCC cells. Furthermore, NEAT1 silence significantly decreased the expression of CNN2, which was the direct target of miR-296-5p. Besides that, the tumor suppression caused by NEAT1 silence could be rescued by CNN2 restoration or miR-296-5p inhibition in vitro. Additionally, NEAT1 indirectly regulated CNN2 expression by competing to miR-296-5p in vitro and in vivo.

Conclusion: LncRNA NEAT1 contributes to HCC progression by regulating miR-296-5p/CNN2 axis, providing a novel regulatory mechanism for HCC development and a promising therapeutic target for the HCC treatment.

Keywords: CNN2; HCC; NEAT1; miR-296-5p; progression.

Figure 1

The expression of NEAT1 and CNN2 is up-regulated in HCC tissues and cells. (A–C) The expression of NEAT1 and CNN2 in HCC and normal tissues was detected using qRT-PCR or Western blot. (D–F) The expression of NEAT1 and CNN2 in normal human hepatocyte THLE-2 and HCC cell lines (HepG2 and Huh7) was measured by qRT-PCR or Western blot. *P<0.05.

Figure 2

NEAT1 silence inhibits cell proliferation, migration and invasion but induces apoptosis in HCC. HepG2 and Huh7 cells were transfected with si-NEAT1 or si-NC. (A) The level of NEAT1 was examined by qRT-PCR. (B, C) Cell proliferation was analyzed by MTT assay. (D) Flow cytometry was used to analyze cell apoptosis. (E, F) The number of migration and invasion cells were detected by transwell assay. *P<0.05.

Figure 3

NEAT1 silence suppresses HCC progression by regulating CNN2 expression. HepG2 and Huh7 cells were transfected with si-NC, si-NEAT1, si-NEAT1 + pcDNA-Control, si-NEAT1 + pcDNA-CNN2. (A, B) The expression of CNN2 was detected using qRT-PCR or Western blot. (C, D) MTT assay was performed to determine cell proliferation. (E) The apoptotic cells were measured using flow cytometry. (F, G) Transwell assay was applied to examine cell migration and invasion abilities. *P<0.05.

Figure 4

NEAT1 silence mediates inhibition on HCC progression by directly sponging miR-296-5p. (A) The binding condition between NEAT1 and miR-296-5p were predicted, and the red bases represented the putative binding sites. (B) The Luciferase activity was analyzed in 293T cells co-transfected with WT-NEAT1 or MUT-NEAT1 and miRNA NC, miR-296-5p mimic. (C) The expression of miR-296-5p in HepG2 and Huh7 cells transfected with si-NC or si-NEAT1 was measured using qRT-PCR. (D, E) The RIP assay was used to determine the interaction between miR-296-5p and NEAT1 in HepG2 and Huh7 cells. (F) The expression of miR-296-5p in HCC tissues and normal tissues was detected using qRT-PCR. (G) The expression of miR-296-5p in normal human hepatocyte THLE-2 and HCC cell lines (HepG2 and Huh7) was measured by qRT-PCR. (H) The expression of miR-296-5p was detected in HepG2 and Huh7 cells after transfection with inhibitor NC or miR-296-5p inhibitor using qRT-PCR. (I, J) Cell proliferation was analyzed by MTT assay. (K) Flow cytometry was performed to measure apoptotic cells. (L, M) The number of migration and invasion cells was analyzed by transwell assay. *P<0.05.

Figure 5

Overexpressed miR-296-5p suppresses HCC progression by directly targeting CNN2. (A) The binding condition between CNN2 and miR-296-5p were predicted, and the red bases were the putative binding sites. (B) The Luciferase activity was analyzed in 293T cells co-transfected with WT-CNN2 or MUT-CNN2 and miRNA NC, miR-296-5p mimic. (C, D) The expression of CNN2 in HepG2 and Huh7 cells transfected with miRNA NC or miR-296-5p mimic was measured using qRT-PCR or Western blot. (E, F) The RIP assay was used to determine the interaction between miR-296-5p and CNN2 in HepG2 and Huh7 cells.(G, H) MTT assay was utilized to determine cell proliferation. (I) The apoptotic cells were examined using flow cytometry. (J, K) Transwell assay was performed to determine cell migration and invasion. *P<0.05.

Figure 6

NEAT1 indirectly regulates CNN2 expression by competing for the binding of miR-296-5p in HCC cells. (A–B) The expression of CNN2 was detected in HepG2 and Huh7 cells transfected with si-NC, si-NEAT1, si-NEAT1 + inhibitor NCor si-NEAT1 + miR-296-5p inhibitor using qRT-PCR or Western blot analysis. *P<0.05.

Figure 7

Knockdown of NEAT1 inhibits tumor growth of HCC in vivo. Huh7 cells stably transfected with sh-NEAT1 was used to establish xenograft models. (A) Tumor volume was calculated every week. (B) Mice were killed and tumor weight was analyzed in each group. (C–F) The levels of NEAT1, miR-296-5p and CNN2 were measured in two groups by qRT-PCR or Western blot. *P<0.05.