LINC01287 regulates tumorigenesis and invasion via miR-298/MYB in hepatocellular carcinoma

Abstract

Recently, it was reported that long non-coding RNAs (lncRNAs) participated in promoting hepatocellular carcinoma (HCC) initiation and progression. Herein, we reported that the expression level of LINC01287 was elevated in HCC cell lines and tissues. LINC01287 down-regulation inhibited HCC cells growth and invasion both in vitro and in vivo. LINC01287 exerted as a ceRNA and negatively regulated miR-298 expression. MYB was identified as a downstream target of miR-298. The miR-298/MYB axis mediated LINC01287's effect on HCC. To the best of our knowledge, our findings provided the first evidence that LINC01287 functioned as an oncogene in HCC. LINC01287 may be a candidate prognostic biomarker and a target for new therapies in HCC patients.

Keywords: LINC01287; MYB; hepatocellular carcinoma; miR-298.

Figure 1

LINC01287 expression level was up‐regulated in hepatocellular carcinoma (HCC) cell lines and tissues. A, LINC01287 expression was significantly increased in primary HCC tissues. B, The expression level of LINC01287 was up‐regulated in advanced stage HCC patients. C, LINC01287 expression was higher in HCC cells when compared with a normal liver cell line. D and E, High‐level expression of LINC01287 was associated with a shorter overall survival and disease‐free survival time of HCC patients (blue and green curves represent low expression and high expression of LINC01287, respectively). *Represents P value < 0.05

Figure 2

LINC01287 inhibition decreased hepatocellular carcinoma (HCC) cell proliferation and invasion. A, LINC01287 expression in Huh‐7 cells transduced with control shRNA vector (sh‐ctrl) or LINC01287 shRNA vector (sh‐LINC01287). B, The MTT assay revealed that LINC01287 down‐regulation significantly decreased cell proliferation. C, Colony formation assay demonstrated that oncogenic survival was significantly decreased in sh‐LINC01287 cells when compared with sh‐ctrl cells. D, sh‐LINC01287 cells displayed a significantly higher frequency of cells at the G1 phase and a lower frequency of cells at S phase. E, LINC01287 down‐regulation affected the expression of G1/S phase checkpoint proteins. F, LINC01287 down‐regulation decreased the HCC cell invasion ability, as revealed by the Boyden assay. G, The expression levels of MMP‐2, MMP‐3 and MMP‐9 protein were lower in sh‐LINC01287 cells. H, Expression level of E‐cadherin increased, while expression levels of N‐cadherin and vimentin were decreased in sh‐LINC01287 cells

Figure 3

LncRNA LINC01287 acted as ceRNA to regulate miR‐298 expression. A, The binding sites of miR‐298 on LINC01287. B, The RIP assay revealed that LINC01287 and miR‐298 were enriched in the same Ago2 immunoprecipitates. C, MiR‐298 expression was increased in sh‐LINC01287 cells when compared with sh‐ctrl cells. D, Cotransfection of miR‐298 and LINC01287‐Wt strongly decreased the luciferase activity, while cotransfection of miR‐ctrl and LINC01287‐Wt did not change the luciferase activity. Cotransfection of miR‐298 and LINC01287‐Mut did not change the luciferase activity either. E, miR‐298 decreased LINC01287 expression, while anti‐miR‐298 increased LINC01287 expression

Figure 4

MYB was a downstream target of miR‐298. A, The binding sites of miR‐298 on MYB. B, The luciferase assay showed that cells transfected with miR‐298 had less luciferase activity than those transfected with miR‐ctrl. C, miR‐298 repressed MYB mRNA expression in hepatocellular carcinoma (HCC) cells. D, miR‐298 repressed MYB protein expression in HCC cells. E, Anti‐miR‐298 treatment led to the restoration of MYB in sh‐LINC01287 cells. F, The MTT assay revealed that sh‐LINC01287 cells grew more slowly than the sh‐ctrl cells, while overexpression of MYB rescued the effect. G, The colony formation assay showed that sh‐LINC01287 cells formed smaller and fewer colonies than the sh‐ctrl cells, which was counteracted by overexpression of MYB. H, LINC01287 down‐regulation affected the cell cycle distribution, which was counteracted by overexpression of MYB. I, LINC01287 down‐regulation inhibited HCC cell invasion ability, which was rescued by overexpression of MYB

Figure 5

LINC01287 inhibition decreased tumour growth and invasion in vivo. A, Compared with sh‐ctrl cell‐derived xenograft tumours, sh‐LINC01287 cell‐derived xenograft tumours grew more slowly. B, The mean weight of sh‐LINC01287 cell‐derived xenograft tumours was significantly less when compared with sh‐ctrl cell‐derived xenograft tumours. C, Knockdown of LINC01287 significantly decreased the percentage of Ki‐67‐positive cells in tumours when compared with the negative control group. D, Knockdown of LINC01287 decreased lung metastasis in vivo