LncRNA UCA1 facilitated cell growth and invasion through the miR-206/CLOCK axis in glioma

Glioma cell lines of U251, U87, SW1783, and LN229, and normal human astrocytes (NHAs) were obtained from Shanghai Bank of Tissues (Shanghai, China). All the cells were maintained in DMEM (Gibco, USA) with 10% FBS (Gibco, USA). They were kept at 37 °C with 5% CO₂. Sixty groups of glioma tissues and adjacent normal tissues were harvested from the Affiliated Hospital of Guizhou Medical University. The normal tissue was obtained from patients with fresh autopsy material (donation from individuals who died in traffic accident and confirmed to be free of any prior pathologically detectable conditions). The tissues were placed in liquid nitrogen. The patients didn’t get any chemotherapy or radiotherapy before surgery, with written informed consents for their tissues. This research was performed with approval from the Ethics Committee board from the Affiliated Hospital of Guizhou Medical University.

qRT-PCR was employed in our experiments to detect the mRNA expressions in cells transfected with various RNAs. Total RNAs were reversely transcripted via PrimeScript RT reagent (Takara, Japan). qRT-PCR was conducted using SYBR Green Master Mix II (Takara, Japan) on ABI7300. The expression of miRNA was measured with reference to U6, and was calibrated with GAPDH. The primer sequences were listed as follows:

UCA1 (forward, 5′-ACGCTAACTGGCACCTTGTT-3′ and reverse, 5′-TGGGGATTACTGGGGTAGGG-3′), GAPDH (forward, 5′-GTCAACGGATTTGGTCTGTATT-3′ and reverse, 5′-AGTCTTCTGGGTGGCAGTGAT-3′), miR-206 (forward, 5′-CGGGCTTGTGGAATGGTAAGC-3′ and reverse, 5′-GCTTCGGCAGCACATATACTAAAAT-3′), and U6 (forward, 5′-CGCTTCACGAATTTGCGTGTCAT-3′ and reverse, 5′-ATGGAACGCTTCACGA-3′).

In order to examine the invasive abilities of glioma cells transfected with related genes, we carried out migration and invasion assays. For migration assay, 10,000 cells in serum-free medium were plated into the upper chamber. Culture medium was put in the lower chamber. After 1 day’s culture, the inserts were fixed and stained by 0.1% crystal violet. The migration ability could be analyzed. For invasion assay, the upper chamber of the inserts was pre-coated with 50 mg/L matrigel. Besides that, all the other procedures were the same.

To fully understand the impact of UCA1, we used lentiviral vector transfections. U251 cells and SW1783 cells were co-transfected to lentivirus plasmid, which were constructed with shRNA of UCA1 sequence. LV-con was the comparison group. Lentiviral vector was added with UCA1 fragment to construct LV-UCA1. MiR-206 mimic or negative control (NC) was transfected via Lipofectamine 2000 (Invitrogen, USA).

Western blotting was utilized in our study to reveal the protein expressions by different RNAs. Proteins were extracted via 10% SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). Antibodies to E-cadherin (1:500, ab40772, abcam), N-cadherin (1:500, ab18203, abcam), vimentin (1:500, ab8978, abcam), CLOCK (1:500, ab201974, abcam) and GAPDH (1:500, ab-8245, abcam) were purchased from Abcam, USA. The reaction was measured by chemiluminescence (Cell Signaling Technology).

To identify the relationship among UCA1, miR-206, and CLOCK, we carried out luciferase reporter assays. UCA1-WT or UCA1-MUT binding miR-206 were sub-cloned to pGL3-Basic vector. MiR-206 mimics were co-transfected with 10 μg pLUC-WT-UCA1 or pLUC-MUT-UCA1. CLOCK-WT or CLOCK-MUT binding miR-206 were sub-cloned to pGL3-Basic vector. MiR-206 mimics were co-transfected to pLUC-WT-CLOCK or pLUC-MUT-CLOCK.

To examine the expressions of E-cadherin, N-cadherin, and vimentin immunofluorescence assays were utilized. Cells were fixed by 4% paraformaldehyde, permeabilized by 0.2% Triton X-100, and blocked by 1.5% normal donkey serum. Primary antibodies were bond for an hour followed by another hour’s incubation with Alexa Fluor 488-linked secondary antibody. The nucleus was stained with DAPI. The fluorescence was measured by a fluorescence microscope (Carl Zeiss, Germany).

To further confirm the effects of UCA1 in glioma, in vivo experiments were performed. Eighteen female nude BALB/c mice aged 6 weeks were recruited. In front dorsum of mice, six were injected by U251 cells (5 × 10⁶/mice), six were administered of U251 cells infected by LV-sh-UCA1 (5 × 10⁶/mice), and six were administered of U251 cells infected by LV-sh-con (5 × 10⁶/mice). We recorded the tumor size every 7 days. After 6 weeks, mice were anesthetized by cervical dislocation. This study was approved by the animal experiment ethics committee of the Affiliated Hospital of Guizhou Medical University and conducted in strict accordance with the national institutes of health guidelines for the care and use of experimental animals.

RNA immunoprecipitation protocol (RIP) was conducted (Millipore, USA). Cells were lysed and incubated by human anti-Ago2 antibody, with magnetic beads (Millipore, USA) or control antibody (Millipore, USA).

SPSS 16.0 and GraphPad Prism were utilized for statistical analysis. The median values were used as cut-off scores to discriminate between low and high expression level. Data were presented as mean ± SD of at least three groups of experiments. One-way ANOVA or Student’s t-test was utilized for comparisons. Fisher’s test was employed to measure the variations between categorical variables. p < 0.05.

Firstly, we aim to reveal the expression profiles of UCA1 in glioma tissues and cell lines by qRT-PCR experiments. Figure 1a displays the relative UCA1 expression in normal tissues (60) and glioma tissues (60). Compared to normal tissues, UCA1 expressions were remarkably up-regulated in glioma tissues (p < 0.001). Figure 1b shows the UCA1 expressions in U251, U87, SW1783, LN229 cells, from which UCA1 was over-expressed compared to NHAs (p < 0.01). From 0 to 60 months, Fig. 1c shows the Kaplan–Meier overall survival curves by different UCA1 levels. Long-term follow-up from this study has substantiated this finding, with survival of 86.96% at 12 months, 60.87% at 24 months, 56.52% at 36 months, 47.83% at 48 months and 30.43% at 60 months for patients with higher UCA1 levels versus 100%, 89.19%, 83.78%, 72.97% and 59.46%, respectively, for patients with lower UCA1 levels. We noticed that patients with higher UCA1 levels had poorer survival compared to those with lower levels.

UCA1 was over-expressed in glioma cells and tissues. Then we hope to know the role of UCA1 in the glioma cell migration and invasion by migration assay and invasion assay. Figure 2a, b shows the transwell assay results transfected with LV-sh-con and LV-sh-UCA1 in U251 and SW1783 (p < 0.01). It revealed that the down-regulation of UCA1 inhibited glioma cell migration and invasion. Figure 2c, d demonstrates the results from western blotting and immunofluorescence. We noticed that the knockdown of UCA1 elevated the expression of epithelial marker E-cadherin, but reduced the expression of N-cadherin and vimentin.

In order to understand the associations between miR-206 and UCA1, we performed online searching about their common sequences, as well as luciferase assay. Figure 3a predicts the common sequence of UCA1 and miR-206. It also shows the luciferase reporter constructs with UCA1-WT (wild-type) or UCA1-MUT (mutant). Figure 3b, c demonstrate the luciferase activity of miR-206 in UCA1-WT and UCA1-MUT, in U251 and SW1783, respectively. It was obvious that the activity of miR-206 was significantly suppressed in UCA1-WT, in both cells (p < 0.01). Figure 3c, e shows the relative expression of IgG and Ago2 in the groups of UCA1 and miR-206, in U251 and SW1783, respectively. RIP assay showed that UCA1 and miR-206 expression were more abundant in Ago2 pellet than in the control IgG pellet (p < 0.01). In addition, Additional file 1: Figure S1 also shows that miR-206 expression was highly up-regulated in cells transfected with sh-UCA1. Additional file 2: Figure S2a, c demonstrates that miR-206 expression was inhibited in glioma cancer cell lines and glioma tissues, in contrast to normal cells and tissues. From these results, the association of UCA1, miR-206, and Ago2 was confirmed, and miR-206 was a target of UCA1 in vitro.

Additional file 2: Figure S2b, d illustrates that CLOCK expression was up-regulated in glioma cancer cell lines and tissues, compared with normal cells and tissues. Figure 4 suggested that CLOCK was a direct target of miR-206. Figure 4a predicts the common sequence between CLOCK and miR-206. It also shows the luciferase reporter with CLOCK-WT (wild-type) and CLOCK-MUT (mutant). Figure 4b, c show the luciferase activity of CLOCK-WT or CLOCK-MUT co-transfected with miR-206 mimics or miR-206 mimics plus LV-UCA1, into U251 cells or SW1783, respectively. The luciferase activity of miR-206 was obviously reduced in CLOCK-WT, compared with CLOCK-MUT (p < 0.01). But this effect was attenuated by UCA1 (observed from the results in miR-206 + LV-UCA1). CLOCK mRNA expression was significantly decreased in glioma cells transfected with miR-206 mimics and LV-shUCA1 while it was increased in LV-UCA1 transfected glioma cells (Fig. 4d, p < 0.01). Consistent with the qRT-PCR results, western blot results indicated the same tendency (Fig. 4e, p < 0.01).

After characterizing the roles of UCA1, miR-206, CLOCK in glioma cells and tissues in vitro, we hope to know whether UCA1 may pose an effect on the glioma growth in vivo. Figure 5a shows the pictures of tumors in LV-sh-con and LV-sh-UCA1. Figure 5b, c displays the tumor growth curve from 0 to 42 days, and the tumor weight at 42 days. Figure 5d. e demonstrates the western blot results in LV-sh-con and LV-sh-UCA1. We noticed that CLOCK expression was suppressed by LV-sh-UCA1 in tumor tissues (p < 0.01). The data confirmed that the growth of glioma tumors was suppressed by the silencing of UCA1 in vivo. Besides, the expression of CLOCK protein was also significantly lowered by the knockdown of UCA1 (p < 0.01). In addition, we also conducted the immunohistochemistry in Additional file 3: Figure S3. We found that expression of Ki-67, a cell proliferation marker, and MMP-9, a cell metastatic marker in xenograft tumor tissue samples were inhibited in LV-sh-UCA1, in contrast with LV-sh-con (Additional file 3: Figure S3a). The same results were observed by immunohistochemistry (Additional file 3: Figure S3b). **p < 0.01. scale bar, 100 µm.