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Long non-coding RNA XIST regulates PTEN expression by sponging miR-181a and promotes hepatocellular carcinoma progression

View Article: PubMed Central - PubMed

ABSTRACT

Background: Tumor metastasis often occurs in hepatocellular carcinoma (HCC) and influences the patient’s prognosis, and microRNAs are reported to play key roles in tumor metastasis. This study was conducted to explore the effect of microRNAs on HCC metastasis.

Methods: The levels of miR-181a in HCC tissues, adjacent tissues, metastatic HCC tissues, and non-metastatic HCC tissues at different stages were determined by qRT-PCR. Effect of miR-181a on the proliferation, invasion, and metastasis of HCC cells was estimated by cell counting kits-8 (CCK-8), wound-healing, and Transwell assays. Software analysis and luciferase assays were used to explore the target gene of miR-181a.

Results: MiR-181a was up-regulated in HCC tissues and its expression level in metastatic HCC tissues was much higher than in non-metastasis samples. PTEN was found to be a target gene of miR-181a. MiR-181a had multiple binding sites with the long non-coding RNA (lncRNA) XIST. The regulation of miR-181a on PTEN was mediated by lncRNA XIST. The proliferation and invasion of cells with siXIST were significantly enhanced compared with those of control cells, while knockdown of miR-181a abolished the enhancing effects.

Conclusions: MiR-181a can promote HCC metastasis by targeting PTEN, which is regulated by lncRNA XIST.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3216-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

MiR-181a regulated the proliferation and invasion of HCC cells. a Relative expression level of miR-181a in HCC cell lines with high or low metastatic capacity and normal liver cells. b The proliferation of HCCLM3 cells with strong metastatic capacity was reduced after knockdown of miR-181a. c The proliferation of Huh7 cells with weak metastatic capacity was increased after the overexpression of miR-181a. d and e Representative micrographs of wound healing assay of HCCLM3 and Huh7 cells stably expressing miR-181a. f The effects of miR-181a overexpression or knockdown on invasion of HCCLM3 or Huh7 cells were analyzed using Transwell assays. HCC, hepatocellular carcinoma. All values are mean ± SD. * vs inhibitor NC, p < 0.05. ** vs inhibitor NC, p < 0.01
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Fig2: MiR-181a regulated the proliferation and invasion of HCC cells. a Relative expression level of miR-181a in HCC cell lines with high or low metastatic capacity and normal liver cells. b The proliferation of HCCLM3 cells with strong metastatic capacity was reduced after knockdown of miR-181a. c The proliferation of Huh7 cells with weak metastatic capacity was increased after the overexpression of miR-181a. d and e Representative micrographs of wound healing assay of HCCLM3 and Huh7 cells stably expressing miR-181a. f The effects of miR-181a overexpression or knockdown on invasion of HCCLM3 or Huh7 cells were analyzed using Transwell assays. HCC, hepatocellular carcinoma. All values are mean ± SD. * vs inhibitor NC, p < 0.05. ** vs inhibitor NC, p < 0.01

Mentions: To investigate the role of miR-181a in the development and progression of HCC, we measured miR-181a expression levels in various HCC cell lines (HCCLM3, HepG2, Hep3B, SMMC-7721, and Huh7) and normal liver cell lines (HL-7702 and L-02). Among these cell lines, HCCLM3 is a poorly differentiated HCC cell line with strong metastatic potential. Huh7 is a well-differentiated HCC cell line with weak metastatic ability. The results showed that in HCC cell lines, the relative expression of miR-181a in HCCLM3 cells was the highest, while the value in Huh7 cells was the lowest (Fig. 2a). HCCLM3 and Huh7cells were selected for further study. MiR-181a inhibitor was used to reduce the level of miR-181a in HCCLM3 cells, and miR-181a level was increased by miR-181a mimics in Huh7 cells. The transfection effect is illustrated in Additional file 3: Figure S1. As shown, the miR-181a inhibitor significantly decreased miR-181a level in HCCLM3 cells compared with the inhibitor NC group, whereas miR-181a mimics observably increased miR-181a level in Huh7 cells compared with the NC mimic group. When the proliferation capacity of HCCLM3 cells after transfection with miR-181a inhibitor for 24, 36, and 48 h was determined, cell growth decreased significantly after transfection with miR-181a inhibitor for 36 and 48 h (Fig. 2b). To verify the relationship between miR-181a and tumor growth, the level of miR-181a in Huh7 cells was increased by transfection with miR-181a mimics and cell viability was determined after 24, 36, and 48 h. Results showed the cell vitality increased after transfection with miR-181a mimics (Fig. 2c). Moreover, a wound-healing assay showed that knockdown of miR-181a drastically suppressed the mobility of HCCLM3 cells compared with control cells, whereas miR-181a overexpression promoted the mobility of Huh7 cells (Fig. 2d and e). Transwell assays demonstrated that knockdown of miR-181a significantly inhibited the invasion of HCCLM3 cells compared with that of the control cells, while miR-181a overexpression promoted the invasion of Huh7cells (Fig. 2f). Results of tumor size showed knockdown of miR-181a significantly reduced the tumor size compared with the control, while overexpression of miR-181a increased tumor size (Fig. 3a and b). Moreover, results of tumor weight also showed that knockdown of miR-181a dramatically reduced the tumor weight compared with the control, while overexpression of miR-181a increased tumor weight (Fig. 3c and d).Fig. 2


Long non-coding RNA XIST regulates PTEN expression by sponging miR-181a and promotes hepatocellular carcinoma progression
MiR-181a regulated the proliferation and invasion of HCC cells. a Relative expression level of miR-181a in HCC cell lines with high or low metastatic capacity and normal liver cells. b The proliferation of HCCLM3 cells with strong metastatic capacity was reduced after knockdown of miR-181a. c The proliferation of Huh7 cells with weak metastatic capacity was increased after the overexpression of miR-181a. d and e Representative micrographs of wound healing assay of HCCLM3 and Huh7 cells stably expressing miR-181a. f The effects of miR-181a overexpression or knockdown on invasion of HCCLM3 or Huh7 cells were analyzed using Transwell assays. HCC, hepatocellular carcinoma. All values are mean ± SD. * vs inhibitor NC, p < 0.05. ** vs inhibitor NC, p < 0.01
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Fig2: MiR-181a regulated the proliferation and invasion of HCC cells. a Relative expression level of miR-181a in HCC cell lines with high or low metastatic capacity and normal liver cells. b The proliferation of HCCLM3 cells with strong metastatic capacity was reduced after knockdown of miR-181a. c The proliferation of Huh7 cells with weak metastatic capacity was increased after the overexpression of miR-181a. d and e Representative micrographs of wound healing assay of HCCLM3 and Huh7 cells stably expressing miR-181a. f The effects of miR-181a overexpression or knockdown on invasion of HCCLM3 or Huh7 cells were analyzed using Transwell assays. HCC, hepatocellular carcinoma. All values are mean ± SD. * vs inhibitor NC, p < 0.05. ** vs inhibitor NC, p < 0.01
Mentions: To investigate the role of miR-181a in the development and progression of HCC, we measured miR-181a expression levels in various HCC cell lines (HCCLM3, HepG2, Hep3B, SMMC-7721, and Huh7) and normal liver cell lines (HL-7702 and L-02). Among these cell lines, HCCLM3 is a poorly differentiated HCC cell line with strong metastatic potential. Huh7 is a well-differentiated HCC cell line with weak metastatic ability. The results showed that in HCC cell lines, the relative expression of miR-181a in HCCLM3 cells was the highest, while the value in Huh7 cells was the lowest (Fig. 2a). HCCLM3 and Huh7cells were selected for further study. MiR-181a inhibitor was used to reduce the level of miR-181a in HCCLM3 cells, and miR-181a level was increased by miR-181a mimics in Huh7 cells. The transfection effect is illustrated in Additional file 3: Figure S1. As shown, the miR-181a inhibitor significantly decreased miR-181a level in HCCLM3 cells compared with the inhibitor NC group, whereas miR-181a mimics observably increased miR-181a level in Huh7 cells compared with the NC mimic group. When the proliferation capacity of HCCLM3 cells after transfection with miR-181a inhibitor for 24, 36, and 48 h was determined, cell growth decreased significantly after transfection with miR-181a inhibitor for 36 and 48 h (Fig. 2b). To verify the relationship between miR-181a and tumor growth, the level of miR-181a in Huh7 cells was increased by transfection with miR-181a mimics and cell viability was determined after 24, 36, and 48 h. Results showed the cell vitality increased after transfection with miR-181a mimics (Fig. 2c). Moreover, a wound-healing assay showed that knockdown of miR-181a drastically suppressed the mobility of HCCLM3 cells compared with control cells, whereas miR-181a overexpression promoted the mobility of Huh7 cells (Fig. 2d and e). Transwell assays demonstrated that knockdown of miR-181a significantly inhibited the invasion of HCCLM3 cells compared with that of the control cells, while miR-181a overexpression promoted the invasion of Huh7cells (Fig. 2f). Results of tumor size showed knockdown of miR-181a significantly reduced the tumor size compared with the control, while overexpression of miR-181a increased tumor size (Fig. 3a and b). Moreover, results of tumor weight also showed that knockdown of miR-181a dramatically reduced the tumor weight compared with the control, while overexpression of miR-181a increased tumor weight (Fig. 3c and d).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Tumor metastasis often occurs in hepatocellular carcinoma (HCC) and influences the patient&rsquo;s prognosis, and microRNAs are reported to play key roles in tumor metastasis. This study was conducted to explore the effect of microRNAs on HCC metastasis.

Methods: The levels of miR-181a in HCC tissues, adjacent tissues, metastatic HCC tissues, and non-metastatic HCC tissues at different stages were determined by qRT-PCR. Effect of miR-181a on the proliferation, invasion, and metastasis of HCC cells was estimated by cell counting kits-8 (CCK-8), wound-healing, and Transwell assays. Software analysis and luciferase assays were used to explore the target gene of miR-181a.

Results: MiR-181a was up-regulated in HCC tissues and its expression level in metastatic HCC tissues was much higher than in non-metastasis samples. PTEN was found to be a target gene of miR-181a. MiR-181a had multiple binding sites with the long non-coding RNA (lncRNA) XIST. The regulation of miR-181a on PTEN was mediated by lncRNA XIST. The proliferation and invasion of cells with siXIST were significantly enhanced compared with those of control cells, while knockdown of miR-181a abolished the enhancing effects.

Conclusions: MiR-181a can promote HCC metastasis by targeting PTEN, which is regulated by lncRNA XIST.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3216-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus