LncRNA-ZNF281 Interacts with miR-539 to Promote Hepatocellular Carcinoma Cell Invasion and Migration

HCC patients and follow-up

This study included 66 HCC patients (40 men and 26 women; 36–68 years, 53.1 ± 6.2 years) who were selected from the 133 HCC cases admitted to The First Affiliated Hospital of Fujian Medical University from February 2011 to June 2014. Ethics Committee of aforementioned hospital approved this study. Inclusion criteria: (1) newly diagnosed HCC cases; (2) with normal functions of other major organs. Exclusion criteria: (1) recurrent HCC; (2) therapies were initiated; (3) multiple disorders were diagnosed. According to AJCC staging system, the 66 HCC patients included 20, 24, and 22 cases at stages II–IV, respectively. According to their medical record, there were 23 cases of HBV-positive, 22 cases of hepatitis-positive, 10 cases of both positive, and 11 cases of both negative. From the day of admission, the 66 HCC patients were monitored for 5 years or until their death to perform survival analysis. The following patients were excluded in survival analysis: (1) patients died of other diseases; (2) patients died of accident; (3) patients lost during follow-up. Informed consent was signed by all patients and controls.

Biopsies and HCC cell lines

Under the guidance of magnetic resonance imaging, liver biopsy was performed to collect both HCC and adjacent (within 2 cm around tumor) nontumor tissue samples. All the tissue samples were subjected to histopathological examinations to confirm that correct samples were harvested.

Two human HCC cell lines SNU-475 and PLHC-1 (ATCC, USA) were included in this study. The cell culture medium was the mixture of 10% fetal bovine serum (FBS) and 90% RPMI-1640 Medium. Cell culture conditions were 37°C, 5% carbon dioxide, and 95% humidity.

Cell transfections

ZNF281 expression plasmid was constructed using pcDNA3.1 vector (Sangon, Shanghai, China). Negative control (NC) small interfering RNA and miR-539 mimic were purchased from RIBOBIO (Guangzhou, China). To perform cell transfections, SNU-475 and PLHC-1 cells were harvested at 70%–80% confluence and lipofectamine 2000 (Sangon) was used to transfect 12 nM vector (empty pcDNA3.1 vector as NC group) or 40 nM miRNA (NC miRNA and NC group) into 3 × 10⁶ cells. Untransfected cells were used as Control (C) cells. Following experiments were performed at 24 h post-transfection.

RNA extractions

SNU-475 and PLHC-1 cells were harvested at 24 h post-transfection and cells were counted. Liver biopsies were ground in liquid nitrogen. RNAzol (Sigma-Aldrich, USA) was used to extract total RNAs from 3 × 10⁵ cells and 0.02 g tissue samples with all operations performed according to the instructions from Sigma-Aldrich. miRNAs were harvested by precipitating RNAs using 85% ethanol.

Quantitative polymerase chain reaction assay

DNase I was used to treat all RNA samples to remove genomic DNAs. MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Lucigen) was used to reverse transcribe RNA samples into computed tomography (cDNA). With cDNA as template, quantitative polymerase chain reaction (qPCR) assays were performed using TB Green Advantage qPCR Premix (Clontech, USA). With 18S rRNA as endogenous control, the expression levels of ZNF281 were normalized.

The expression levels of mature miR-539 were also measured. The addition of poly (A), miRNA reverse transcription, and miRNA qPCR assays were all performed using All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia).

All Ct values were processed using 2^−ΔΔCT method. All PCRs were performed in triplicate manner.

Dual-luciferase assay

The interaction between miR-539 and ZNF281 was analyzed by dual-luciferase assay. In this assay, psiCHECK-1 vector (Promega) instead of pcDNA3.1 vector was used. Cells were transfected with either ZNF281 vector + NC miRNA or ZNF281 vector + miR-539. Dual-Luciferase Reporter Assay (Promega) was used to normalize renilla luminescence by firefly luminescence.

Cell invasion and migration analysis

SNU-475 and PLHC-1 cells were harvested at 24 h post-transfection and cells were counted. After that 1 mL serum-free RPMI-1640 Medium was used to resuspend cell pellets containing 3 × 10⁴ cells to make cell suspensions (single cell). To perform transwell assays, the upper chamber was filled with cell suspensions, whereas the lower chamber was filled with a mixture of 20% FBS and 80% RPMI-1640 Medium. It is worth noting that, before invasion assay, upper chamber membranes were coated with Matrigel (250 mg/mL; Millipore, USA) for 12 h at 37°C. Under aforementioned conditions, cells were cultivated for 12 h, following by using crystal violet (0.1%, Sigma-Aldrich) to stain the lower face of the membranes for 20 min at room temperature. Finally, a light microscope was used to observe and count invading and migrating cells.

Statistical analysis

Mean values of three replicates were calculated and used in the following statistical analysis. Correlations were analyzed by Pearson's correlation coefficient. Differences between two types of tissues or two groups were analyzed by paired or unpaired t-test, respectively. Differences among multiple groups were analyzed by analysis of variance (one-way) and Tukey test. With the median expression levels of ZNF281/miR-539 as cutoff values, the 66 patients were grouped into high- and low-level groups (n = 33). Survival curves were plotted using K–M plotter and compared by log-rank test. p-Value <0.05 was statistically significant.

ZNF281 and miR-539 were inversely correlated in HCC

Levels of ZNF281 and miR-539 expression in two types of tissues (HCC and nontumor) were measured by performing qPCR. Expression data were compared between 2 groups by performed paired t-test. It was observed that expression levels of ZNF281 were significantly higher in HCC tissues than in nontumor tissues (Fig. 1A, p < 0.05). In contrast, expression levels of miR-539 were significantly lower in HCC tissues than in nontumor tissues (Fig. 1B, p < 0.05). The correlations between ZNF281 and miR-539 across HCC tissues (Fig. 1C) and nontumor tissues (Fig. 1D) were analyzed by performing Pearson' correlation coefficient. It was observed that ZNF281 and miR-539 were inversely and significantly correlated in HCC tissues but not in nontumor tissues.

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FIG. 1.

ZNF281 and miR-539 were inversely correlated in HCC. Levels of ZNF281 and miR-539 expression in two types of tissues (HCC and nontumor) were measured by performing qPCR. Expression data were compared between 2 groups by performed paired t-test (A). The relationship between miR-539 and ZNF281 (B). The correlations between ZNF281 and miR-539 across HCC tissues (C) and nontumor tissues (D) were analyzed by performing Pearson’ correlation coefficient. *p < 0.05. Triangles represent the data points in the figure. HCC, hepatocellular carcinoma; qPCR, quantitative polymerase chain reaction.

Altered expression levels of ZNF281 and miR-539 predicted poor survival

Survival curves were plotted and compared using aforementioned methods. Comparing with patients in low ZNF281 level groups, patients in high ZNF281 level groups showed significantly lower overall survival (Fig. 2A). However, the overall survival rate of patients in low miR-539 group was significantly lower than that in high-level group (Fig. 2B).

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FIG. 2.

Altered expression levels of ZNF281 and miR-539 predicted poor survival. With the median expression levels of ZNF281 (A)/miR-539 (B) as cutoff values, the 66 patients were grouped into high- and low-level groups (n = 33). Survival curves were plotted using K–M plotter and compared by log-rank test.

ZNF281 and miR-539 downregulated each other in HCC cells

Dual-luciferase assay was performed to analyze the interactions between miR-539 and ZNF281 in both SNU-475 and PLHC-1 cells. Comparing with cells transfected with ZNF281 vector + NC miRNA (NC group), significantly higher luciferase activity was observed in cells transfected with ZNF281 vector + miR-539 (miR-539 group) (Fig. 3A, p < 0.05). To further analyze the relationship between these two factors, SNU-475 and PLHC-1 cells were transfected with ZNF281 expression vector and miR-539 mimic. Comparing with C and NC groups, expression levels of ZNF281 and miR-539 were significantly increased at 24 h post-transfection (Fig. 3B, p < 0.05). Comparing with two controls, ZNF281 overexpression led to the downregulation of miR-539 (Fig. 3C, p < 0.05). In addition, miR-539 overexpression also led to the downregulation of ZNF281 (Fig. 3D, p < 0.05).

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FIG. 3.

ZNF281 and miR-539 downregulated each other in HCC cells. Dual-luciferase assay was performed to analyze the interactions between miR-539 and ZNF281 in both SNU-475 and PLHC-1 cells. Comparing with cells transfected with ZNF281 vector + NC miRNA (NC group), significantly higher luciferase activity was observed in cells transfected with ZNF281 vector + miR-539 (miR-539 group) (A). To further analyze the relationship between these two factors, SNU-475 and PLHC-1 cells were transfected with ZNF281 expression vector and miR-539 mimic. Overexpression of ZNF281 and miR-539 was confirmed by qPCR at 24 h post-transfection (B). The effects of ZNF281 overexpression on miR-539 (C) and the effects of miR-539 overexpression on ZNF281 were also analyzed by qPCR. Experiments were repeated three times and mean values were presented. *p < 0.05. HCC, hepatocellular carcinoma; miRNA, microRNA; NC, negative control; qPCR, quantitative polymerase chain reaction.

The interaction between ZNF281 and miR-539 regulated the invasion and migration of HCC cells

The effects of ZNF281 and miR-539 overexpression on the invasion (Fig. 4A) and migration (Fig. 4B) of HCC cells were analyzed by performing transwell assays. Comparing with C and NC groups, ZNF281 overexpression led to enhanced and miR-539 overexpression led to suppressed HCC cell invasion and migration. In addition, miR-539 overexpression attenuated the effects of ZNF281 overexpression.

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FIG. 4.

The interaction between ZNF281 and miR-539 regulated the invasion and migration of HCC cells. The effects of ZNF281 and miR-539 overexpression on the invasion (A) and migration (B) of HCC cells were analyzed by performing transwell assays. Experiments were repeated three times and mean values were presented. *p < 0.05. HCC, hepatocellular carcinoma.